ES2623288T3 - Use of glycolide hydrolase 61 family proteins in cellulose processing - Google Patents

Abstract

A composition comprising: a) a recombinantly produced GH61 protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2 or fragments of SEQ ID NO: 2 that have GH61 activity; and b) cellulase enzymes including at least one endoglucanase (EG), at least one β-glucosidase (BGL), at least one Type 1 cellobiohydrolase (CBH1), and at least one Type 2 cellobiohydrolase (CBH2).

Description

Use of glycolide hydrolase 61 family proteins in cellulose processing

Field of the Invention

The disclosure generally refers to the field of glycolytic enzymes and their use. More specifically, it provides GH61 proteins from Myceliophtora thermophila, and the use of such proteins in the production of fermentable sugars and cellulosic biomass ethanol.

Background

Cellulosic biomass is a significant renewable resource for the generation of fermentable sugars. These sugars can be used for fermentation and other metabolic processes to produce biofuels, chemical compounds and other commercially valuable end products. While the fermentation of sugars such as glucose to ethanol is relatively simple, the efficient conversion of cellulosic biomass to fermentable sugars is a challenge. Ladisch et al., 1983, Enzyme Microb. Technol 5:82

The conversion of cellulosic biomass to fermentable sugars can begin with chemical, mechanical, enzymatic pre-treatments or other pre-treatments to increase the susceptibility of cellulose to hydrolysis. Such pre-treatment can be followed by enzymatic conversion of cellulose to cellobiose, cello-oligosaccharides, glucose and other sugars and sugar polymers, using enzymes that break cellulose. These enzymes are collectively referred to as "cellulases" and include endoglucanases, β-glucosidases and cellobiohydrolases.

WO 2009/033071 discloses SEQ ID NO: 44 which shares 100% identity with GH61 Myceliophtora thermophila enzyme of SEQ ID: 2 as disclosed herein.

WO 2009/085935 discloses two GH61 enzymes of Myceliophtora thermophila (SEQ ID NO: 2 and 4) and enzymatic mixtures.

Summary of the Invention

The invention provides a composition: a) a recombinantly produced GH61 protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2 or fragments of SEQ ID NO: 2 that have GH61 activity; and b) cellulase enzymes that include at least one endoglucanase (EG), at least one β-glucosidase (BGL), and at least one Type 1 cellobiohydrolase (CBH1), and at least one Type 2 cellobiohydrolase (CBH2).

The invention also provides a method for producing fermentable sugars of cellulosic biomass which comprises contacting a cellulosic substrate with a composition, culture medium or cell lysate containing a GH61 protein and cellulase enzymes, wherein: a) the GH61 protein comprises a amino acid sequence that is at least 80% identical to SEQ ID NO: 2 or a biologically active fragment thereof; and b) cellulase enzymes include at least one endoglucanase (EG), at least one β-glucosidase (BGL), and at least one Type 1 cellobiohydrolase (CBH1), and at least one Type 2 cellobiohydrolase (CBH2).

The invention further provides a method for producing a final product from a cellulosic substrate comprising: a) contacting the cellulosic substrate with a composition of the invention whereby fermentable sugars are produced from the substrate; and b) contacting fermentable sugars with a microorganism in a fermentation to produce a final product.

The invention also provides a method for producing a final product from a cellulosic substrate, comprising: a) obtaining fermentable sugars produced in accordance with a method of the invention; and b) contacting the fermentable sugars with a microorganism in a fermentation to produce a final product.

Disclosure Summary

Here, recombinant proteins of the glycoside Hydrolase 61 (GH61) family obtained from Myceliophtora thermophila, and nucleic acids encoding such proteins are disclosed. The disclosure also provides isolated and purified GH61 proteins from M. thermophila culture broth. These proteins can be used to increase the production of reaction products where a cellulose-containing substrate undergoes saccharification by one or a combination of cellulase enzymes, such as endogluconases, β-glucosidases and cellobiohydrolases. The addition or presence of recombinant or isolated GH61 protein can increase the production of cellulase enzymes, at least 20%, 30%, 50%, 70%, double, triple or more.

The disclosure provides a composition comprising an isolated or recombinant GH61 protein, and a method of preparing such a composition. The protein may be isolated from M. thermophila, or it may be obtained from recombinant production. Amino acid sequences of twenty-four GH61 M proteins are provided.

full length thermophila GH61. The disclosed GH61 proteins include proteins that comprise an amino acid sequence that is at least about 60%, about 70%, about 80%, about 85%, about 90%, about 92%, about 93%, about 94%, about 95 %, approximately 96%, approximately 97%, approximately 98% or approximately 99% identical to any one of the listed proteins (SEQ ID NOS: 1-30) or a fragments thereof biologically active. An exemplary GH61 protein is GH61a (SEQ ID NO: 2). Other exemplary GH61 proteins include GH61o, GH61v, GH61x, GH61b and GH61e (SEQ ID Nos: 6, 13, 15, 16, 19, 30). Other exemplary GH61 proteins include GH61f, GH61v, GH61p, GH61g and GH61i (SEQ ID Nos: 7, 13, 20, 21, 23, 26).

In one aspect of the disclosure, the composition prepared according to the method of this invention comprises an isolated or recombinant GH61 protein and one or more cellulase enzymes listed in this disclosure, including but not limited to cellulose enzymes selected from endoglucanases. (EG), βglucosidases (BGL), Type 1 cellobiohydrolases (CBH1), and Type 2 cellobiohydrolases (CBH2).

Another embodiment of the disclosure is a method to produce a fermentable sugar from cellulosic substrate. A suspension comprising the substrate contacts a composition comprising a GH61 protein to produce fermentable sugars such as glucose and xylose from the substrate. The composition may also contain one or more enzymes selected from cellulose proteins (endoglucanases, β-glucosidases, Type 1 cellobyohirolases, and Type 2 cellobiohirolases), esterases, xylanases, hemicellulases, lipases, proteases, amylases and glucoamylases. The substrate can be derived from, for example, wheat, wheat straw, sorghum, corn, rice, barley, sugarcane bagasse, herbs, sticky grass, corn grain, corn cobs, corn fiber or a combination of themselves, exemplified by pre-treated corn straw.

Fermentable sugar can be recovered and used to produce a final product such as an alcohol (such as ethanol or butanol), a sugar alcohol (such as sorbitol), an organic acid (such as lactic acid, acrylic acid, acetic acid, succinic acid, glutamic acid , citric acid or propinoic acid), an amino acid (such as glycine, lysine) or aspartic acid, an organic acid, an alkane, an alkene, a diol or glycerol.

Another embodiment of the disclosure is a method for increasing the production of fermentable sugars in a saccharification reaction by one or more cellulose enzymes, performing the reaction in the presence of a GH61 protein as referred to above.

In another aspect, the disclosure provides a method for hydrolyzing a cellulose substrate. The substrate contacts a composition comprising one or more recombinant GH61 proteins, one or more glucosidases (BGL) and one or more cellobiohydrolases (CBH). In some embodiments of the disclosure, the enzyme composition is substantially free of endoglucanase (EG). Hydrolysis can result in a production of glucose that is at least 20%, 30%, 50%, 70%, double or triple or more than the production of the same reaction performed in the absence of said GH61 protein.

Another embodiment of the disclosure is a method of producing a final product from a cellulosic substrate. The substrate contacts a composition containing GH61 protein as already referred to under conditions under which fermentable sugars are produced. The fermentable sugars then contact a microorganism in a fermentation to produce a final product as listed above. This method is suitable for preparing an alcohol, particularly ethanol, where the microorganism is a yeast.

Other embodiments of the disclosure include: a) the aforementioned GH61 recombinant proteins, optionally produced with a heterologous signal peptide; b) a nucleic acid sequence encoding the GH61 protein that may be operably linked to a heterologous promoter; c) a host cell that produces such recombinant GH61 proteins, exemplified by M. thermophila, yeast, a Chaetomium, Thielavia, Acremonium, Myceliophthora, Aspergillus, or Trichoderma host cells.

The disclosure further incorporates the use of a recombinantly produced GH61 protein in ethanol production. The GH61 protein comprises an amino acid sequence that is at least 60%, approximately 70%, approximately 80%, approximately 85%, approximately 90%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96% , approximately 97%, approximately 98% or approximately 99% identical to any one of the listed proteins or a fragment thereof having GH61 activity.

Here we describe a nucleic acid that encodes a GH61 protein. The disclosure also provides a cell that contains a recombinant nucleic acid sequence encoding a protein sequence of this invention (SEQ ID Nos: 1 to 30) operably linked to a heterologous promoter. The host cells can be (for example) M. thermophila cells or yeast cells. The recombinant nucleic acid sequence may include SEQ ID NO. 31 or any one of SEQ ID Nos: 32 to 59. The cell can also express at least one recombinant cellulase protein selected from an endoglucanase (EG), a β-glucosidase (BGL), a Type 1 cellobiohydrolase (CBH1), and / or a Type 2 cellobiohydrolases (CBH2) and / or variants of said cellulase proteins.

In one aspect, the invention provides a composition containing a GH61 protein (eg, SEQ ID NO: 2), an endoglucanase (EG), a β-glucosidase (BGL), a Type 1 cellobiohydrolase (CBH1), and / or a Type 2 cellobiohydrolase (CBH2), where the combined mass of the GH61, EG, BGL, CHB1 and CBH2 protein is at least about 20%, 40%, 60%, 70%, 80%, 90%, 95% or substantially all of the total cell-free protein in the composition. One, two, three or all four of the enzymes CBH1, CBH2, EG and BGL that may be present in the composition may be variants derived from naturally occurring cellulase proteins. The composition may also be a cellulose culture broth containing cellulase proteins.

Here a GH61 protein is described which comprises any of SEQ ID Nos: 3 to 30 or a secreted fragment thereof. Optionally, the protein comprises the secreted fragment and the corresponding signal peptide sequence, if present. In a related aspect the disclosure provides a variant of GH61 protein with at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at minus about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequential identity with a polypeptide or a biologically active fragment provided herein.

Here we describe a recombinant nucleic acid sequence encoding a protein described above, which can have the sequence of the naturally occurring gene or a fragment thereof. The sequence encoding the nucleic acid protein may be operably linked to a heterologous signal sequence. Typically, the recombinant nucleic acid sequence is operably linked to a heterologous promoter. A host cell containing a recombinant nucleic acid of the disclosure is disclosed. The cell can express at least one, two, three or four recombinant cellulose proteins selected from endoglucanases (EG), β-glucosidases (BGL), Type 1 cellobiohydrolases (CBH1), and / or Type 2 cellobiohydrolases (CBH2).

Here a composition is described that contains at least one isolated protein comprising a sequence selected from the secreted part of SEQ ID Nos. 1 to 30, and / or at least one biologically active fragment thereof. The composition may also contain at least one endoglucanase (EG), a β-glucosidase (BGL), a Type 1 cellobiohydrolase (CBH1), and / or a Type 2 cellobiohydrolase (CBH2), where the combined mass of the GH61 protein , EG, BGL, CHB1 and CBH2 is at least about 70% of the total cell-free protein in the composition.

In one aspect, the disclosure provides methods for saccharification (a) by culturing a cell of the invention under conditions where at least one GH61 protein is secreted in the culture broth, and (b) combining the broth and a cellulosic biomass under conditions where it occurs. saccharification, where (a) can take place before or simultaneously with (b).

In one aspect, the disclosure provides a method for hydrolyzing a starch, which comprises contacting the starch with a composition comprising one or more recombinant GH61 proteins and one or more amylase (s).

Description of the drawings

FIGURE 1 is taken from an experiment using recombinant GH61 protein produced from Myceliophtora thermophila. The protein was tested to improve cellulase activity using cellulase enzymes from a M. thermophila culture broth. FIGURE 1 (A) shows the improved production percentage of a saccharification reaction performed in the presence of GH61a, compared to the same reaction in the absence of GH61a. In FIGURE 1 (B), the data is plotted to show total glucose production.

FIGURE 2 is taken from an experiment using GH61 containing isolated fractions of M. thermophila culture broth. The GH61, GH61f, GH61p and / or GH61a proteins were combined with CBH1a and CBH2. These results demonstrate that an enzyme mixture comprising these components has sufficient enzymatic activity for the conversion of a cellulose substrate to glucose.

FIGURE 3 shows the effect of the GH61a protein on the viscosity of cellulosic biomass.

Detailed description

introduction

The filamentous fungus Myceliophtora thermophila was determined to produce GH61 proteins. GH61 proteins increase the production of fermentable sugars when a cellulose-containing substrate undergoes saccharification by one or more cellulase enzymes. Fermentable sugars produced by saccharification can

used, among other uses, in fermentation reactions to produce final products, such as, but not limited to, ethanol.

GH61 proteins can be isolated from M. thermophila cells. GH61 proteins can also be produced recombinantly by expressing a nucleic acid encoding any of the GH61 protein sequences provided in the disclosure, including wild type sequences and wild type sequence fragments.

The preparation and use of GH61 proteins, and the compositions of the invention are described in more detail in the sections below.

II. Definitions

Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, fermentation, microbiology and related fields, which are known to those skilled in the art. Unless otherwise defined herein, all the technical and scientific terms used here have the same meaning as commonly understood by one skilled in the art to which this invention pertains. Although some methods and materials similar or equivalent to those described herein may be used in practice or in the tests of the present invention, preferred methods and materials are described. In fact, it is intended that the present invention is not limited to the particular methodology, protocols and reagents described herein, since these may vary depending on the context in which they are used. The titles provided here are not limitations of the various aspects or accomplishments.

However, in order to facilitate the compression of the present invention, a number of terms are defined below. The numerical ranges include the numbers that define the range. Thus, each numerical range disclosed herein is intended to include each narrowest numerical range that is classified within a broader numerical range, as if such narrower numerical ranges were expressly written here. It is also intended that each maximum (or minimum) numerical limitation disclosed herein includes each lowest (or highest) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

As used herein, the terms "comprising (n)" and their cognates are used in their inclusive sense (that is, equivalent to the term "including (n)" and their corresponding cognates).

As used herein and in the appended claims, the singular "one", "one", "the", "the" includes the plural reference unless the context clearly dictates otherwise. Thus, for example, the reference to a "host cell" includes a plurality of such host cells.

Unless otherwise indicated, nucleic acids are written from left to right in a 5 ’to 3’ orientation; The amino acid sequences are written from left to right in carboxy orientation, respectively. The titles provided here are not limitations of the various aspects or embodiments that may be taken by reference to the specification as a whole. Therefore, the terms defined below are more fully defined by reference to the specification as a whole.

As used herein, a "GH61 protein" is a protein with GH61 activity (or "cellulase enhancer"). A "GH61 protein) or a GH61 polypeptide may have a sequence of a naturally occurring (wild-type) protein or may comprise variations relative to a wild-type protein.

As used herein, the GH61 protein sequences shown in Tables 1 and 2 (SEQ ID Nos: 1-30) are considered wild type sequences.

A protein has "GH61 activity" or "cellulose-promoting activity" or is "biologically active" if, when included in a saccharification reaction (for example, it is performed using an endogluconase, a β-glucosidase and cellobiohydrolases Type 1 and Type 2 ) results in a greater amount (that is, greater production) of one or more soluble sugars (eg glucose) than the saccharification reaction performed under the same conditions in the absence of the GH61 protein. A "biologically active variant" is a variant or fragment that retains at least some (for example, at least 10%) of the GH61 activity of the wild-type protein.

As used herein, a "variant" GH61 protein (or polynucleotide encoding a GH61 protein) is a GH61 protein that comprises one or more modifications related to wild-type GH61 (or the wild-type polynucleotide encoding GH61). Modifications include substitutions, insertions, deletions and / or truncation of amino or carboxy of one or more of the amino acid residues (or of one or more nucleotides or codons in the polynucleotide). A variant comprising a relative elimination of wild-type protein can be referred to as a "fragment."

As used herein, a "fragment" of a GH61 protein is (a) a polypeptide with a wild-type sequence but comprising a deletion relative to SEQ ID Nos: 1-30 or (b) a variant of GH61 comprising a removal relative to a polypeptide of SEQ ID Nos: 1-30.

A "substitution" of amino acid in a protein sequence is a substitution of a single amino acid with a sequence with another amino acid.

An amino acid substitution may be a "conservative" substitution, in which case the substituted amino acid shares one or more chemical properties with the amino acid it replaces. Shared properties include the following: basic amino acids: arginine (R), lysine (K), histidine (H); acidic amino acids: glutamic acid (E) and aspartic acid (D); uncharged polar amino acids: glutamine (Q) and asparagine (N); hydrophobic: leucine (L), isoleucine (I), valine (V); aromatic amino acids: phenylalanine (F), tryptophan (W), and tyrosine (Y); sulfur-containing amino acids: cysteine (C), methionine (M); Small amino acids: glycine (G), alanine (A), serine (S), threonine (T), proline (P), cysteine (C), and methionine (M).

The term "pre-protein" has a standard meaning in the art and refers to a polypeptide that includes a linked amino terminal signal peptide region (or leader sequence). The signal peptide is split from the preprotein by a signal peptidase simultaneous to protein secretion. The secreted part of the protein can be referred to as "mature" protein or "secreted" protein. Thus, an amino acid sequence of a preprotein will comprise a signal peptide part and a secreted (mature) part.

As used herein, the terms "signal peptide has its usual meaning and refers to an amino acid sequence linked to the amino terminus of a polypeptide, which directs the encoded polypeptide to a cell secretory sequence.

"Saccharification" refers to an enzyme-catalyzed reaction that results in hydrolysis of a complex carbohydrate to fermentable sugars (eg, monosaccharides such as glucose or xylose). The enzymes can be cellulose enzymes, such as endogluconases, β-glucosidases and cellobiohydrolases of Type 1 and Type 2, and combinations of such cellulase enzymes. Cellulase enzymes can be from a culture broth of a wild-type organism or constructed with cellulase that produces cellulase enzymes (for example, a fungus such as

M. thermophila or yeast).

The "hydrolysis" of cellulose or other polysaccharide (eg, starch) occurs when glycosidic bonds between at least some of the adjacent monosaccharides hydrolyze, thus separating the previously bound monomer pairs.

"Increasing" the production of a product (such as a fermentable sugar) from a reaction occurs when a particular component present during a reaction (such as a GH61 protein) causes more than one product to be produced, compared to a reaction performed under them. conditions with the same substrate and other substituents, but in the absence of the component of interest.

The terms "improved" or "improved properties", as used in the context of describing the properties of a GH61 variant, refer to a GH61 variant polypeptide that shows an improvement in a property or properties compared to Wild type GH61 (for example, SEQ ID NO: 2) or a specified reference polypeptide. Improved properties may include, but are not limited to, greater protein expression, greater thermoactivity, greater thermostability, greater specific activity, greater substrate specificity, greater resistance to the substrate or inhibition of the final product, greater chemical stability, reduced glucose inhibition, increased resistance to inhibitors (for example, acetic acid, lectins, tannic acids and phenolic compounds) and an altered pH / temperature profile.

The term "cellulase" (or "cellulase enzyme") refers in general terms to enzymes that catalyze the hydrolysis of β-1,4-glycosidic cellulose bonds to shorter chains of cellulose, oligosaccharides, cellobiose and / or glucose, for example, endoglucanases, β-glucosidases and cellobiohydrolases.

As used herein, the term "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides in a one-chain or double-chain form, and complements thereof.

As used herein, a "gene" is a nucleic acid sequence that encodes a protein. The nucleic acid may or may not have introns, may or may not be recombinant and may or may not further comprise elements that affect transcription or translation. For the purposes of this description, a cDNA sequence encoding a protein can sometimes be referred to as a "gene."

The terms "recombinant nucleic acid" have their conventional meaning. A recombinant nucleic acid, or equivalently, "polynucleotide," is one that is inserted into a heterologous location such that it is not associated with nucleotide sequences that would normally flank the nucleic acid.

when found in nature (for example, a nucleic acid inserted into a vector or a genome of a heterologous organism). In the same way, a nucleic acid sequence that does not appear in nature, for example, a variant of a naturally occurring gene, is recombinant. A cell that contains a recombinant nucleic acid, or protein expressed in vitro or in vivo of a recombinant nucleic acid are also recombinant. Examples of recombinant nucleic acids include a DNA sequence that encodes a protein that (i) is operably linked to a heterologous promoter and / or (ii) encodes a fusion polypeptide with a protein sequence and a heterologous signal peptide sequence.

Nucleic acids "hybridize" when they are associated, typically in solution. Nucleic acids hybridize due to a variety of well-characterized physicochemical forces, such as hydrogen bonding, solvent exclusion, base accumulation and the like. As used herein, the terms "severe hybridization wash conditions" in the context of nucleic acid hybridization experiments, such as Southern and Northern hybridization, are sequence dependent, and are different under different environmental parameters. An extensive guide for nucleic acid hybridization is found in Tijssen, 1993 "Laboratory techniques in biochemistry and molecular biology - Hybridization with nucleic acid probes", Part I, Chapter 2 (Elsevier, New York). For polynucleotides of at least 100 nucleotides in length, conditions of low to very high severity are defined as follows: pre-hybridization and hybridization at 42 ° C in 5xSSPE, 0.3% SDS, 200 µg / ml of sperm DNA of cut and denatured salmon and 25% formamide for low severity, 35% formamide medium and medium-high severity, or 50% formamide for high and very high severity, following standard Southern test procedures. For polynucleotides of at least 100 nucleotides in length, the carrier material is finally washed three times every 15 minutes using 2xSSC, 0.2% SDS 50 ° C (low severity), at 55 ° C (medium severity), at 60 ° C (medium severity -high), at 65 ºC (high severity) or at 70 ºC (very high severity).

The terms "expression vector" refer to a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of the disclosure, and that is operably linked to additional segments that provide for its transcription (eg, a promoter, a transcription killing sequence, enhancers) and optionally a selectable marker.

For purposes of this disclosure, a promoter is "heterologous" to a gene sequence if the promoter is not naturally associated with the gene. A signal peptide is "heterologous" to a protein sequence when the signal peptide sequence is not associated with the protein by nature.

In relation to regulatory sequences (eg, promoters), the terms "operably linked" refer to a configuration where a regulatory sequence is located in a position relative to a polypeptide coding sequence such that the regulatory sequence influence the expression of the polypeptide. In relation to a signal sequence, the terms "operably linked" refer to a configuration where the signal sequence encoding an amino-terminal signal peptide is fused with the polypeptide, such that gene expression produce a pre-protein.

As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs.

The terms "biomass", "biomass substrate", "cellulosic biomass", cellulosic raw material "," lignocellulosic raw material "and" cellulosic substrate "all refer to materials containing cellulose. For simplicity, the terms "cellulosic substrate" herein are used to refer to cellulose-containing materials that can function with celluloses (with optionally present GH61 proteins), typically after pre-treatment, to produce fermentable sugars. Examples of cellulosic substrates include, but are not limited to, biomass such as wood, wood pulp, paper pulp, corn fiber, corn grain, corn cobs, grain residues such as corn husks, corn stubbles, herbs , wheat, wheat straw, barley, barley straw, hay, rice straw; rod grass, leftover paper, leftover paper and pulp processing, woody and herbaceous plants, fruit and vegetable pulp, distiller grain, rice pod, cotton, hemp, flax, sisal, sugarcane bagasse, sorghum, soybeans , components obtained from grinding grains, trees, branches, roots, leaves, wood chips, sawdust, thickets and shrubs, vegetables, fruits and flowers and mixtures thereof. In some embodiments, the cellulosic material is "pre-treated", or treated using methods known in the art, such as chemical pre-treatment (eg, pre-treatment with ammonia, pre-treatment with dilute acid, pre-treatment with alkali diluted or solvent exposure), physical pre-treatment (for example, exposure to lightning or radiation), mechanical pre-treatment (for example, spraying or grinding) and biological pre-treatment (for example, application of microorganisms that solubilize lignin) and combinations thereof, to increase the susceptibility of cellulose to hydrolysis. Cellulosic substrates and their processes are described in more detail below.

A cellulosic substrate is “derived from” a specific source (such as corn or wheat) by a process that involves obtaining the source or a physical part of the source (such as a corn cob or wheat straw), and then optionally pre -treat the source or part.

A cellulase protein sequence (that is, a cellulose variant) is "derived from" a cellulase sequence when produced by introducing variations in a wild-type sequence using in vitro mutagenesis

or molecular evolution methods. Typically, the protein sequence will be at least 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95% to the sequence wild type

It is said that a cellulosic substrate is "pre-treated" when it has been processed by some physical and / or chemical means to facilitate saccharification.

"Fermentable sugars" refer to simple sugars (monosaccharides, disaccharide and short oligosaccharides) such as, but not limited to, glucose, xylose, galactose, arabinose, mannose and sucrose. Fermentable sugar is any sugar that a microorganism can use or ferment.

The terms "transform" or "transformation," as used in reference to a cell, mean that a cell has a non-native nucleic acid sequence integrated into its genome or as an episome (eg, plasmid) that is maintained through of multiple generations.

The term "introduced", as used in the context of nucleic acid sequence insertion into a cell, means conjugated, transfected, transduced or transformed (collectively "transformed") or otherwise incorporated into the genome, or maintained as a Episome in a cell.

A "cellulose constructed" cell is a cell that comprises at least one, at least two, at least three or at least four recombinant sequences encoding a cellulase or cellulase variant, and where the expression of the cellulase (s) ) or variant (s) of cellulase has been modified in relation to the wild type form. The expression of a cellulase is "modified" when a cellulase variant that occurs in an unnatural way is expressed or when a cellulase that occurs naturally is overexpressed. One way to overexpress a cellulase is to operatively link a strong promoter (optionally constitutive) with the cellulase encoding the sequence. Another way to overexpress a cellulase is to increase the copy number of a heterologous, variant or endogenous cellulase gene. The cell constructed by cellulase can be a fungal cell, such as a yeast cell or a filamentous fungal cell (for example, Acidothermus cellulolyticus, Thermobifida fusca, Humicola grisea, Myceliophthora thermophila, Chaetomium thermophilum, Acremonium sp., Thielavia sp, Trichoderma rei, Trichoderma rei Aspergillus sp.). In some embodiments, the cell constructed by cellulase is an M. thermophila cell.

The term "cultivate" refers to growing a population of microbial cells (eg, fungal) under a liquid or solid culture medium. Some cultured cells express or secrete proteins, such as GH61 proteins or cellulase proteins. When cells grow in a liquid medium, proteins can be secreted into the medium, which is referred to by several terms, including cell culture medium, cell culture supernatant and culture broth.

As used herein, the term "recovery" refers to the harvesting, isolation, collection and separation of a protein, sugar, cell or final product (eg alcohol) from a culture broth or, alternatively, culture medium solid.

As used herein, the term "isolated" refers to a nucleic acid, polypeptide or other component that is partially or completely separated from components with which it is normally associated by nature (such as other proteins, nucleic acids or cells).

A product that has been "purified" is a product that has been enriched from the source from which it is obtained at least 10 times, and optionally at least 100 times, or at least 1000 times. For example, a protein that is obtained from a culture broth where it represents 0.1% of the total protein can be purified to be 1%, 2.5%, 10%, 25%, 50%, 80%, 95% or 100% (w / w) of the protein in the preparation. A product that has been "partially purified" has been enriched from the source from which it is produced at least 2 times, at least 5 times, but still contains at least 50%, and sometimes at least 90% (p / p) of unrelated components other than the solvent.

"Fractioning" a liquid product as a culture broth means applying a separation process (such as salt precipitin, column chromatography, size exclusion and filtration) or a combination of such processes to obtain a solution where a protein desires (such as GH61 protein, a cellulase enzyme or a combination of them) has a higher percentage of total protein in the solution than the initial liquid product.

A "suspension" is an aqueous solution where one or more solid components are dispersed, such as cellulosic substrate.

As used herein, a "composition" comprising one or more proteins may be, for example, a cell-free composition comprising the protein (s), a cell lysate, a culture broth comprising the (s) protein (s), for example, in secreted form; a cell comprising the protein (s), such as a recombinant cell that expresses the protein (s); a mixture of two or more cell populations that express different proteins (for example, a cell that expresses a recombinant GH61 protein and a second cell that expresses cellulase protein).

The terms "cell free composition" refer to a composition that contains proteins where cells and cell debris have been removed, such as a mixture of purified cellulase or a cell incubation broth containing secreted protein, from which cells have been removed. and insoluble material.

The terms "percentage identity", "% identity", "identical percentage" and "identical%" are used interchangeably to refer to a comparison of two optimally aligned sequences on a comparison window. The comparison window may include additions or deletions in any sequence to optimize alignment. The percent identity is the number of positions that are identical between the sequences, divided by the total number of positions in the comparison advantage (including positions where one of the sequences has a space). For example, a protein with an amino acid sequence that coincides at position 310 with a GH61a sequence (which has 323 amino acids in the secreted form) will have 310/323 = 95.9% identity with the reference. Similarly, a protein variant that has 300 residues (that is, less than the full length) and matches the reference sequence in 280 positions will have 280/300 = 93.3% identity. While optimal alignment and classification can be done manually, the process can be facilitated using a computer-implemented alignment algorithm. Examples are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402. Alternatively, the degree of identity between two amino acid sequences can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-53), which has been implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000, Trends in Genetics 16: 276-77), preferably version 3.0 or later. The optional parameters used are an open space penalty of 10, a space extension penalty of 0.5, and the replacement matrix EBLOSUM62 (EMBOSS version of BLOSUM62). The production of “longer identity” with the Needle label (obtained using the nobrief option) is used as the percentage identity and is calculated as follows: (Identical Waste x 100) / (Alignment Length - Total Number of Spaces in alignment).

III. GH61 gene identification

Twenty-four endogenous GH61 proteins to Myceliophthora thermophila were identified as described in Example 1. See TABLE 1 and TABLE 2 below. As shown in Examples 2 and 3, a particular GH61 protein of M. thermophila with the designation GH61a (SEQ ID NO: 2) was shown to improve saccharification reactions in the presence of cellulases. Similarly, other GH61 proteins (SEQ ID Nos. 3 to 30) can be used to improve cellulase activity.

TABLE 1 provides the sequence of a GH61 pre-protein (SEQ ID NO: 1), which shows the predicted native signal peptide underlined, and the predicted (mature) secreted form (SEQ ID NO: 2). The sequence ID NO: 2 is 323 amino acids in length. It is contemplated that certain GH61a protein variants will comprise residues 11-323 of SEQ ID NO: 2 (including, but not limited to, truncated amino-terminal fragments), or will have at least: about 70%, about 75%, about 80% , approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98% or approximately 99% sequence identity with residues 11-323 of SEQ ID NO: 2. In some embodiments, the GH61 protein variant will have at least 90% identity with residues 11-323 of SEQ ID NO: 2 and will have 315 residues in length.

TABLE 2 provides 28 GH61 pre-protein sequences, with the predicted native signal peptide underlined. GH61t and GH61n are not predicted to have signal peptides. Except where otherwise specified

or be clear from the context, the reference to one or more sequences listed in TABLE 2 is inserted to encompass one or more of the pre-protein form and the secreted form (that is, a protein comprising the entire sequence, and a protein that does not include the underlined sequence). It is also contemplated that certain GH61 proteins comprise residues 11 with terminal C of the sequences shown in Table 2 (including, but not limited to, truncated amino-terminal fragments), or will have at least: about 70%, about 75% , approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98% or approximately 99% identity sequence with residues 11 for terminal C of these sequences. In Table 2 the following denominations can be used: B = SEQ ID NO. full length protein (including signal peptide); C = Signal Peptide (Underline); D = Protein

mature (not underlined); E = Part of D that begins in residue 11 of the secreted part (that is, between space and terminal C, a truncated part of amino terminal).

The signal peptide limits in TABLE 1 and TABLE 2 are predicted based on the analysis of the primary sequence. It is possible that the actual division site differs from the predicted site by up to several residues (for example, 1-20, for example up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). The presence of a heterologous signal peptide can also affect the site of division. A signal peptide division can be determined by expressing the full length sequence and identifying amino terminal residues of the secreted GH61 protein. It is expected that the addition or removal of various residues in the amino terminal of the mature protein has little or no effect on the activity of the secreted protein.

TABLE 1

Glycoside hydrolase 61 GH61a protein

SEQ ID NO:

Designation

one

GH61a Pe-Protein:

2

GH61a Mature Protein:

35 TABLE 2 Glycoside hydrolase 61 GH61a protein

B

Designation Sequence (Putative Signal Peptide Sequence)

3

GH61l

4

GH61m

5

GH61n

6

GH61o

7

GH61p

8

GH61q

9

GH61 r

(keep going)

10

GH61s

eleven

GH61t

12

GH61u

13

GH61v

14

GH61w

fifteen

GH61x

16

GH61b

17

GH61c

18

GH61d

19

GH61e

twenty

GH61f

twenty-one

GH61g

22

GH61h

2. 3

GH61i

24

GH61j

25

GH61k

26

GH61p2

27

GH61q2

28

GH61r2

29

GH61t2

30

GH61e2

IV. Recombinant nucleic acids and proteins

In one aspect, the disclosure provides recombinant nucleic acids comprising sequences of

60 proteins set forth in TABLE 1 or TABLE 2 and variants (for example, biologically active variants) thereof. The disclosure also provides expression vectors containing recombinant nucleic acids, cells comprising a recombinant nucleic acid or vector, recombinant proteins produced by such cells and methods for using cells and proteins.

In one aspect, the disclosure provides a recombinant nucleic acid sequence encoding a pre-protein comprising SEQ ID NO: 1-30, the mature (secreted) protein encoded in SEQ ID NO: 1-30 or a truncated amino terminal fragment of SEQ ID NO: 1-30.

In one aspect, the disclosure provides a recombinant, isolated or purified GH61 protein having a sequence comprising SEQ ID NO: 1-30, the mature (secreted) protein encoded in SEQ ID NO: 1-30 or a truncated amino terminal fragment of SEQ ID NO: 1-30.

In one aspect, the disclosure provides a recombinant nucleic acid sequence encoding a GH61 protein with at least about 70%, at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92% , approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98% or approximately 99% sequence identity with a sequence comprising SEQ ID NO: 1-30, the mature protein (secreted ) encoded in SEQ ID NO: 1-30 or an amino terminal truncated fragment of SEQ ID NO: 1-30. In a related aspect, the disclosure provides a recombinant, isolated or purified GH61 protein that has a sequence as set forth above. In some cases, a conservative amino acid substitution may be preferred over other types of substitutions.

GH61 proteins can be endogenous proteins isolated from fungal cells (eg, M. thermophila) or can be produced recombinantly.

As discussed in more detail below, GH61 proteins may comprise an endogenous or heterologous signal peptide. As discussed in more detail below, a nucleic acid encoding a GH61 protein can be operably linked to a promoter, such as a heterologous promoter.

In one embodiment, the disclosure provides a recombinant nucleic acid sequence comprising a sequence selected from SEQ ID NOS: 31-59.

The nucleic acid sequence used to express a GH61 protein may be a native sequence obtained from M. thermophila encoding the respective protein, or a part thereof encoding the mature protein. GH61 specimens encoding M. thermophila sequences are shown in SEQ ID NOS: 31 to 59. Alternatively, nucleic acid sequences encoding a specific protein may be designated by reference to the genetic code. In some embodiments, a sequence that can be codon optimized for a host cell other than M. thermophila (for example, yeast cells) is used. See GCG CodonPreference, Genetics Computer Group Wisconsin Package; Codon W, John Peder, University of Nottingham; McInerney, J. O., 1998, Bioinformatics 14: 372-73, in which case the nucleic acid sequence is different from the sequence that occurs naturally.

In some embodiments, the GH61 proteins of the disclosure are biologically active, that is, they have GH61 activity. GH61 activity can be measured using methods known in the art, including methods described below. Polypeptides that lack GH61 activity can also find a variety of uses, including use for the generation of antibodies for purification of GH61 proteins. Unless clarified from the context, the reference here to a GH61 protein (including variants) refers to proteins with GH61 activity.

It is preferred that GH61 proteins that are variants of SEQ ID NOS: 1-30 have at least 10% of the activity of the same molar amount of the wild-type protein from which they are derived. They can have at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99 % of the activity of the wild-type protein.

GH61 proteins that are of variants of SEQ ID NOS: 1-30 may be shorter than wild-type protein by approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 60%, approximately 70 % or about 80% compared to the reference sequence (wild type) and / or part of a fusion protein in which a part of GH61 protein binds to one or more sequences. These variants may have at least about 70%, at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96 %, about 97%, about 98% or about 99% sequence identity with a sequence comprising SEQ ID NO: 1-30, the mature (secreted) protein encoded in SEQ ID NO: 1-30 or an amino truncated fragment SEQ ID NO terminal: 1-30. GH61 proteins can have an internal elimination or an amino or carboxy terminal elimination in relation to a wild type sequence.

GH61 proteins may comprise an amino terminal and / or carboxy terminal elimination and / or internal elimination, but where the rest of the amino acid sequence is at least about 60%, 70%, 75%, 80%, 85%, 90% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding positions in the sequence with which it is being compared (for example, a GH61 variant of length full disclosure). In some embodiments, the chitin binding domain, or GH61 domain is removed. In some embodiments, the protein conserves substantially all of the activity of the full length polypeptide.

A mature GH61 protein can have at least 70%, 80%, 90% or 95% sequence identity with a wild type sequence, and has substantially full length (at least 90% of the length of the wild type sequence).

The disclosure also provides a recombinant GH61 nucleic acid sequence and expressed protein thereof, where the protein has the sequence of GH61f, GH61a, GH61v, GH61p, GH61g and GH61i (SEQ ID Nos: 2, 7, 13, 20, 21 , 23, 26), or a secreted fragment thereof; as well as variants at least about 70%, at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96% , about 97%, about 98% or about 99% identical to any of GH61f, GH61v, GH61p, GH61g and GH61i (SEQ ID Nos: 2, 13, 20, 21, 23, 26); or any one of GH61a, GH61o, GH61v, GH61x, GH61b and GH61e (SEQ ID Nos: 2, 6, 13, 15, 16, 19, 30), fragments and variants thereof, and nucleic acids encoding such proteins. The disclosure also includes expression vectors comprising any one of the aforementioned nucleic acid sequences, cells comprising such expression vectors and isolated GH61 proteins produced by the cells. Preferably, the GH61 variant protein has cellulase enhancing activity. The protein encoding the sequences may include a heterologous signal peptide and / or may be operably linked to a heterologous promoter.

Without claiming to be linked to a particular mechanism of action, in the presence of GH61, the hydrolysis of sugar polymers (for example, cellulose substrates) by enzymes produces more product for a particular period of time, proceeds more quickly or completes more fast when GH61 protein is present, compared to a similar reaction under the same conditions where GH61 protein is absent.

The GH61 proteins of the disclosure having cellulose enhancing activity can be identified using standard methods for mapping function in a polypeptide, as is known in the art. For example, a truncated variant can be expressed and then tested in a GH61 activity assay. Additional truncation can be introduced until activity is lost, at which point the minimum functional unit of the protein could be identified. Fragments that contain any part of the protein below the identified size will typically be functional, as would be the fusion constructs that contain at least the functional core of the protein.

To generate biologically active variants that incorporate one or more amino acid changes in a sequence encoding GH61 (any of SEQ ID Nos: 1 to 30), substitutions may be introduced in the protein sequence and the expressed protein for activity retention.

A random or semi-random mutation strategy can be used to generate a large collection of active variants. The standard texts PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al., Eds.) And MOLECULAR CLONING: A LABORATORY MANUAL (Sambrook et al., Eds.) Describe techniques that employ chemical mutagenesis, cassette mutagenesis, oligonucleotide degeneration, mutual preparation of oligonucleotides, linker scan mutagenesis, alanine scan mutagenesis and error prone PCR. Other efficient methods include Stratagene E. coli mutant strains (Greener et al., Methods Mol. Biol. 57: 375, 1996) and the Maxygen DNA mixing technique (Patten et al., Curr. Opin. Biotechnol .8: 724, 1997; Harayama, Trends Biotechnol. 16:76, 1998; US Patents 5,605,793 and 6,132,970). To increase the variation, a technology that generates more abrupt changes can be used as a DNA mixing technique.

Commercially available kits may be used to obtain variants, including the GeneTailor ™ Site Directed Mutagenesis System sold by InVitrogen ™ Life Technologies; the BD Diversify ™ Random Mutagenesis Kit, sold by BD Biosciences / Clontech; Template Generation System ™, sold by MJ Research Inc., the mutant strain of E. coli XL1-Red ™, sold by Stratagene; and the GeneMorph® Random Mutagenesis kit, also sold by Stratagene. By employing any of these systems together with a suitable GH61 activity assay, variants can be generated and tested in a manner with high performance.

Alternatively or in addition to, the user may employ a directed evolution strategy. See, for example, U.S. Patent 7,981,614; Methods for generating polynucleotides that have the desired characteristics; US 2011/0034342: Method to generate an optimized and diverse population of variants; US Patent 7,795,030: Methods and compositions for cellular and metabolic engineering; US Patent 7,647,184: High evolutionary performance directed by rational mutagenesis; United States Patent

6,939,689: Re-assembly of exonuclease-mediated nucleic acid in directed evolution; and United States Patent

United 6,773,900: Final selection in directed evolution. Mutagenesis can be performed according to any of the techniques known in the art, including randomized and site-specific mutagenesis. Directed evolution can be performed with any of the techniques known in the art to investigate the production of variants including mixing.

The methods of mutagenesis and directed evolution are well known in the art. See U.S. Patents Nos. 5,605,793, 5,830,721, 6,132,970, 6,420,175, 6,277,638, 6,365,408, 6,602,986, 7,288,375, 6,287,861, 6,297,053, 6,576,467 , 6,444,468, 5,1811238, 6,117,679, 6,165,793, 6,180,406, 6,291,242, 6,995,017, 6,395,547, 6,506,602, 6,519,065, 6,506,603, 6,413,774, 6,573,098 , 6,323,030, 6,344,356, 6,372,497, 7,868,138, 5,834,252, 5,928,905, 6,489,146, 6,096,548, 6,387,702, 6,391,552, 6,358,742, 6,482,647, 6,335 .160, 6,653,072, 6,355,484, 6,03,344, 6,319,713, 6,613,514, 6,455,253, 6,579,678, 6,586,182, 6,406,855. 6,946,296, 7,534,564, 7,776,598, 5,837,458, 6,391,640, 6,309,883, 7,105,297, 7,795,030, 6,326,204, 6,251,674, 6,716,631, 6,528,311, 6,287. 862, 6,335,198, 6,352,859, 6,379,964, 7,148,054, 7,629,170, 7,620,500, 6,365,377, 6,358,740, 6,406,910, 6,413,745, 6,436,675, 6,961,664, 7,430,477, 7,873,499, 7,702,464, 7,783,428, 7,747,391, 7,747,393, 7,751,986, 6,376,246, 6,426,224, 6,423,542, 6,479,652, 6,319,714, 6,521. 453, 6,368,861, 7,421,347, 7,058,515, 7,024,312, 7,620,502, 7,853,410, 7,957,912, 7,904,249, and all related counterparts that are not from the United States; Ling et al., Anal. Biochem., 254 (2): 157-78 [1997]; Dale et al., Meth. Mol. Biol., 57: 369-74 [1996]; Smith, Ann. Rev. Genet., 19: 423-462 [1985]; Botstein et al., Science, 229: 1193-1201 [1985]; Carter, Biochem. J., 237: 1-7 [1986]; Kramer et al., Cell, 38: 879-887 [1984]; Wells et al., Gene, 34: 315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3: 284-290 [1999]; Christians et al., Nat. Biotechnol., 17: 259-264 [1999]; Crameri et al., Nature, 391: 288-291 [1998]; Crameri et al., Nat. Biotechnol., 15: 436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 94: 4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14: 315-319 [1996]; Stemmer, Nature, 370: 389-391 [1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91: 10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336.

In some embodiments, a GH61 protein of the disclosure has an amino acid sequence that is encoded by a nucleic acid that hybridizes under severe conditions (i.e., high, high or very high average severity) with the complement of SEQ ID NO: 31 -59 and includes GH61 activity.

V. GH61 Activity Tests

The GH61 protein cellulase enhancing activity of the disclosure can be determined using any suitable GH61 activity assay. For example, a purified or recombinant GH61 protein is obtained from this disclosure and then analyzed for GH61 activity by combining it with cellulase enzymes in a saccharification reaction, and it is determined whether there is an increase in glucose production, compared to the same reaction. of saccharification performed without the GH61.

In one technique, GH61 activity can be analyzed by combining cellulosic substrate with cellulase enzymes (for example, 5-10 mg of total cellulase enzyme weight per gram of substrate) in the presence and absence of GH61 protein. In some embodiments, cellulase enzymes are a defined set of recombinant cellulase enzymes of M. thermophila.

In another technique, the broth of a wild-type M. thermophila culture (with or without the complementation of the GH61 protein) is used. GH61 activity is demonstrated by increased glucose production in the presence of exogenous GH61 (that is, beyond any resulting improvement of endogenous GH61 in the broth).

It is also possible to use a broth supplemented with one or more purified enzymes.

Suitable enzymes include isolated recombinant enzymes cloned from M. thermophila, such as endogluconase (EG), β-glucosidase (BGL); Type 1 cellobiodrilase (CBH1) and / or Type 2 celloiodiodollase (CBH2) in any combination suitable for the substrate chosen to produce a measurable product. Exemplary cellulase enzymes that can be used to analyze GH61 activity can have amino acid sequences selected from any of SEQ ID Nos: 61 to 68.

In an exemplary assay to measure GH61 activity of M. thermophila derived from GH61 proteins and variant proteins, the cellulase enzymes used are M. thermophila BGL1 (SEQ ID NO: 66; Badhan et al., Bioresour Technol. 2007 Feb; 98 ( 3): 504-10); M. thermophila CBH1 (SEQ ID NO: 67; Park JI et al., Badhan et al., Bioresour Technol. 2007 Feb; 98 (3): 504-10); and M. thermophila CBH2 (SEQ ID NO: 68). In some embodiments, endoglucanase is also used: M. thermophila EG2 (SEQ ID NO: 65; Rosgaard L. et al., Prog. 2006; 22 (2): 493-8; Badhan et al., Supra).

Alternatively, commercially available preparations comprising a mixture of cellulase enzymes, such as Laminex ™ and Spezyme ™ from Genencor International, Rohament ™ from Rohm GmbH, and Celluzyme ™, Cereflo ™ and Ultraflo ™ from Novozymes Inc. can be used.

Cellulose enzyme assays are typically done at 50 ° C, but can also be done at 35, 45, 55, 60 or 65 ° C. The GH61 protein and enzymes are combined with the substrate and incubated to produce fermentable sugars. The sugars are then recovered and quantified to produce glucose. A suitable substrate is wheat straw (for example, pre-treated wheat straw). Other cellulosic substrates listed in this disclosure can be used as an alternative, including corn stubble pre-treated with sulfuric acid (see U.S. Patent 7,868,227) and other substrates described in Section XII below.

An assay method provided by Harris et al., 2010, Biochemistry 49: 33053316 can also be used. In this method, the corn stubble is pretreated with sulfuric acid, washed, incubated with cellulose enzymes and GH61 for several days, and then the production of sugars is quantified by refraction.

Another test method is provided in U.S. Patent No. 7,868,227. In this method, the cellulosic substrate is PCS (corn stubble pre-treated with heat and dilute sulfuric acid; WO 2005/074647); A cellulose enzyme mixture is Cellulcast®, a mixture of cellulose enzymes from the Trichoderma reesei fungus, available from Sigma-Aldrich. PCS hydrolysis is performed in a total reaction volume of 1.0 mL and a PCS concentration of 50 mg / mL in 1 mM manganese sulfate, 50 mM sodium acetate pH buffer

5.0. The test protein is combined with the base cellulase mixture in relative concentrations between 0 and 100% total protein. The protein composition is incubated with PCS at 65 ° C for 7 days. Combined glucose and cellobiose production can be measured by refractive index detection.

GH61 activity is calculated as an increase in glucose production from the substrate by the cellulase (s) in the presence of GH61 protein, compared to the same reaction mixture in the absence of GH61 protein. Typically, the increase is dose dependent in at least a triple range of concentrations. GH61 activity can be expressed as a degree of "synergy" as discussed in Example 8.

The addition or presence of recombinant or isolated GH61 protein can increase the production of the product from cellulase enzymes, for example, at least 1%, at least 5%, at least 10%, at least 20%, 30%, 50 %, 70%, double, triple or more.

SAW. GH61 protein expression

Cell culture, recombinant genetics, protein engineering and fermentation techniques that can be employed in the expression, production and use of GH61 proteins, compositions and other products of this disclosure are known in the art. For convenience, certain aspects are briefly described below. Although described primarily in the context of expression of GH61 proteins, it will be appreciated that the same methods, cells, etc., can be used to express cellulase proteins and other proteins as, but not limited to, those described herein elsewhere.

In some embodiments of the disclosure, the construct further comprises regulatory sequences, for example, a promoter, operably linked to the protein encoding the sequence. Those skilled in the art know large numbers of suitable vectors and promoters.

Signal peptide

In some embodiments of the disclosure, a GH61 protein may include a signal peptide, so that when expressed in a host cell, the mature form (for example, SEQ ID NO: 2) is secreted in a cell culture broth. The GH61 protein (or variant) may include its corresponding native signal peptide as shown in TABLES 1 and 2. Alternatively, a recombinant nucleic acid sequence encoding a protein comprising SEQ ID NO: 2, or the secreted part of any one of SEQ ID Nos: 3 to 30, a truncated amino terminal portion of any one of SEQ ID Nos: 2 to 30, or a variant thereof having a heterologous signal peptide fused with terminal N.

Several signal peptides can be used, depending on the host cell and other factors. Useful signal peptides for filamentous fungal host cells include signal peptides obtained from Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, Lipainosalala Humosala landala lugosala, Lipainosalala humosala landala lugosala, lipa Humosala insolens, lipain Humosala insolens, lipain Humosala insolens, lipain Humosala insolens, lipain Humosala insolens, lipain Humosala insolens, lipain Humosala insolens, lipain Humosala insolens, lipain Humosala landalane and T. reesei cellobiohydrolase II (TrCBH2).

Useful signal peptides for bacterial host cells are the signal peptides obtained from the genes for Bacillus malthogenic amylase NCIB 11837, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, proteases neut proteases Bacillus stearothermophilus (nprT, nprS, nprM), and prsA Bacillus subtilis. More signal peptides are described in Simonen and Palva, 1993, Microbiol Rev 57: 109-137.

Useful signal peptides for yeast host cells also include those of the Saccharomyces cerevisiae alpha-factor genes, SUC2 Saccharomyces cerevisiae invertase (see Taussig and Carlson,

1983, Nucleic Acids Res 11: 1943-54; SwissPro, Access No. P00724), and others. Romans et al., 1992, Yeast 8: 423

488. Variants of these signal peptides and other signal peptides are suitable.

The disclosure also provides recombinant proteins that comprise a signal peptide shown in Table 1 or Table 2 fused with an amino terminal of a heterologous protein (that is, a protein with which it is not associated by nature, which may be a protein other than a GH61 protein). Thus, the signal peptide shown in Table 1 and 2 can be used to cause secretion of a recombinant heterologous protein expressed in a host cell. In some embodiments the host cell is Myceliophthora thermophila.

Promoters

In order to obtain high levels of expression in a particular cell it is often useful to express the GH61 variant of the present disclosure under the control of a heterologous promoter. A promoter sequence can be operatively linked to the 5 'region of the sequence encoding GH61 using routine methods.

Examples of promoters useful for expression of GH61s include fungal promoters. In some embodiments, a promoter sequence that drives the expression of a different gene to a GH61 gene in a fungal strain can be used. As a non-limiting example, a fungal promoter of a gene encoding an endoglucanase can be used. In some embodiments, a promoter sequence that conducts the expression of a GH61 gene in a fungal strain different from the fungal strain from which the GH61 variant was derived can be used. As a non-limiting example, if the GH61 variant is derived from C1, a T. reesei GH61 gene promoter or a promoter such as that described in WO 2010107303 can be used, such as, but not limited to the sequences identified as SEQ ID NO: 25 , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29 in WO 2010107303.

Examples of other suitable promoters useful for directing the transcription of the nucleotide constructs of the present disclosure into a filamentous fungal host cell are promoters obtained from genes for TAKA amylase from Aspergillus oryzae, aspartic proteinase from Rhizomucor miehei, neutral alpha-amylase from Aspergillus niger, Aspergillus niger stable alpha-amylase acid, Aspergillus niger glucoamylase or Aspergillus awamori (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae isossase, aspermidase trypsidase, trypermidase, trypermidase, trypermidase, trypermidase, trypermidase, aspergillus, aspergidase, trypside type of Fusarium oxysporum (WO 96/00787), as well as the promoter NA2-tpi (a hybrid of promoters of the genes for neutral alpha-amylase of Aspergillus niger and triosa phosphate isomerase of Aspergillus oryzae) promotes as cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2, xyn1, amy, and glaA (Nunberg et al., 1984, Mol. Cell Biol., 4: 2306-2315, Boel et al., 1984, EMBO J. 3: 158185 and EPA 137280), and mutant, truncated and hybrid promoters thereof. In a yeast host, useful promoters may be from the genes of Saccharomyces cerevisiae (eno-1) enolase, Saccharomyces cerevisiae galactokinase (gal1), alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae (ADH2 / GAP) , and S. cerevisiae 3-phosphoglycerate kinase. Other promoters useful for yeast host cells are described in Romanos et al., 1992, Yeast 8: 423-488. Promoters associated with chitinase production in fungi can also be used. See, for example, Blaiseau and Lafay, 1992, Gene 120243-248 (filamentous fungus Aphanocladium album); Limon et al., 1995, Curr. Genet, 28: 478-83 (Trichoderma harzianum).

Known promoters for gene control expression in prokaryotic or eukaryotic cells or viruses and which can be used in some embodiments of the invention include SV40 promoter. E. coli lac or trp promoter, phage lambda PL promoter, tac promoter, T7 promoter and the like. In bacterial host cells, suitable promoters include promoters obtained from E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucransa gene (sacB), Bacillus licheniformis (amyl) α-amylase gene, amylase gene Bacillus stearothermophilus (amyM), Bacillus amyloliquefaciens (amyQ) α-amylase gene, Bacillus subtilis xy / A and xy / B genes and β-lactamase prokaryotic gene.

Any other promoter sequence that conducts expression in a suitable host cell can be used. Suitable promoter sequences can be identified using well known methods. In one technique, a putative promoter sequence binds to a 5 ′ sequence that encodes a reporter protein, the construct is transfected into the host cell (e.g., C1) and the reporter's expression level is measured. Reporter expression can be determined by measuring, for example, mRNA levels of the reporter sequence, an enzymatic activity of the reporter protein or the amount of reporter protein produced. For example, promoter activity can be determined using the green fluorescent protein as a coding sequence (Henriksen et al., 1999, Microbiology 145: 729-34) or a lacZ reporter gene (Punt et al., 1997, Gene, 197: 189- 93). Functional promoters can be derived from promoter sequences that occur naturally by directed evolutionary methods. See, for example, Wright et al., 1005, Human Gene Therapy, 16: 881-892.

Additional promoters include those of M. thermophila, provided in U.S. Patent Application No. 13 / 214,406 filed on August 22, 2010, as well as in WO 2010/107303.

Vectors

The present invention makes use of recombinant constructs comprising a sequence encoding GH61 as described above. The nucleic acid constructs of the present disclosure comprise a vector, such as a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (CAB), an artificial yeast chromosome (CAL) and the like, where a nucleic acid sequence of the disclosure. The polynucleotides of the present disclosure can be incorporated into any one of a variety of expression vectors suitable for expressing a polypeptide. Suitable vectors include chromosome, non-chromosome and synthetic DNA sequences, for example, derivatives of SV40; bacterial plasmids, phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccine virus, adenovirus, avian smallpox virus, pseudorrabia, adenovirus, adeno-associated virus, retrovirus and the like. Any vector that converts genetic material into a cell and, if replication is desired, that is replicable and viable in the relevant host can be used.

The disclosure provides expression vectors to cause a GH61 protein to be produced from a suitable host cell, which may be a fungus (eg, M. thermophila or yeast). Any vecotr of, but not limited to, derived from viral vectors can be selected; bacterial plasmids, phage DNA; baculovirus, yeast plasmids; vectors derived from combinations of plasmids and phage DNA, and recombinant shuttle vectors. The vector can be introduced into a host cell with a polynucleotide encoding GH61 so that it is operably linked to a promoter that is active in the host cell. The vector is selected to express the encoded protein, and can replicate as an episome in the planned host cell, or integrate into the genome of the host cell.

In a particular aspect the present disclosure provides an expression vector comprising a GH61 polynucleotide operably linked to a heterologous promoter. The expression vectors of the present disclosure can be used to transform an appropriate host cell to allow the host to express the GH61 protein. Methods for recombinant protein expression in fungi and other organisms are well known in the art, and a number of expression vectors are available or can be constructed using routine methods. See, for example, Tkacz and Lange, 2004, ADVANCES IN FUNGAL BIOTECHNOLOGY FOR INDUSTRY, AGRICULTURE, AND MEDICINE, KLUWER ACADEMIC / PLENUM UBLISHERS. NY; Zhu et al., 2009, Construction of two input vectors for gene expression in fungal plasmid. 6: 128-33; Kavanagh, K. 2005, FUNGI: BIOLOGY AND APPLICATIONS Wiley.

Host cells

The GH61 proteins of the disclosure can be expressed in a host cell comprising a recombinant nucleic acid encoding the GH61 protein. The host cell can also express other proteins of interest, particularly one or more cellulase enzymes that work in concert with the GH61 protein in the saccharification process. In one embodiment the host cell is a cell constructed with cellulase. Thus, cellulase enzymes can be expressed endogenously by the host cell, or they can be expressed from other nucleic acids.

In another technique, two or more host cell populations, each expressing a different protein

or a set of proteins (for example, a GH61 protein and a cellulase) can be grown together. The two host cells can be the same or can be of different cell species. Cells expressing GH61 protein and cells expressing cellulase enzymes can be combined and cultured together to produce compositions of this invention that contain both GH61 proteins and cellulase enzymes. Alternatively, the culture broth of each cell population can be collected separately, optionally fractionated to enrich the respective activities and then mixed to produce the desired combination.

Fungal host cells include, but are not limited to Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, Fungi imperfecti. In some embodiments, the preferred fungal host cells are yeast cells, and filamentous fungal cells, including all filamentous forms of the Eumycotina and Oomycota subdivision. Hawksworth et al., In Ainsworth and Bisby’s DICTIONARY OF THE FUNGI, 8th edition, 1995, CAB International, University Press, Cambridge, UK. Filamentous fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose and other complex polysaccharides, and are morphologically distinct from yeast. Trichoderma can also be a Source of one or more cellulases for use in combination with GH61 proteins.

The host cell can be of a species of Achlya, Acremonium, Aspergillus, Aureobasidium, Azospirillum, Bjerkandera, Cellulomonas, Cephalosporium, Ceriporiopsis, Chrysosporium, Clostridium, Coccidioides, Cochliobolus, Coprinus, Coriolus, Corynascoccus, Corynasia, Corynasia, Corynasia, Corynasia , Endothia, Erwinia, Escherichia, Fusarium, Gibberella, Gliocladium, Gluconacetobacter, Humicola, Hypocrea, Kuraishia, Mucor, Myceliophthora, Neurospora, Nicotiana, Paenibacillus, Penicillium, Periconia, Phaeosphaeria, Phlebiaporavomorom, Pyromyces, Pyromyces, Pyromyces. , Rhizopus, Ruminococcus, Saccharomycopsis, Salmonella, Schizophyllum, Scytalidium, Septoria, Sporotrichum, Streptomyces, Talaromyces, Thermoanaerobacter, Thermoascus, Thermotoga, Thielavia, Tolypocladium, Trametes, Trichoderma, Troeum, Trochumum

Uromyces, Verticillium, Volvariella, Wickerhamomyces, or telemorphs or anamorphs, and synonyms and taxonomic equivalents thereof.

An exemplary host cell is yeast. Examples are Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowia. Yeast may be Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, finlandica Pichia, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, salictaria Pichia, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

An exemplary cell can be Myceliophthora thermophila, sometimes referred to as "C1". As used herein, the term "C1" refers to Myceliophthora thermophila, including a fungal strain described by Garg (See, Garg, Mycopathol., 30: 3-4 [1966]). As used herein, "Chrysosporium lucknowensequot; includes the strains described in United States patents Numbers: 6,015,707, 5,811,381 and 6,573,086; United States Patent Publications Numbers: 2007/0238155, US 2008/0194005, US 2009/0099079; International Patent Publications Numbers: WO 2008/073914 and WO 98/15633, and include, without limitation Chrysosporium lucknowense Garg 27K, VKM-F 3500 D (Access No. VKM F-3500-D), C1 strain UV13-6 (Access No. VKM F-3632 D), C1 strain NG7C-19 (Access No. VKM F3633 D), and C1 strain UV18-25 (VKM F-3631 D), all deposited in the Russian Microorganism Collection of the Russian Academy of Sciences (VKM ), Bakhurhina St. 8, Moscow, Russia, 113184, and any derivative thereof. Although initially described as Chrysosporium lucknowense, C1 can now be considered a strain of Myceliophthora thermophila. Other strains of C1 include cells deposited under accession number ATCC 44006, CBS (Centraalbureau voor Schimmelcultures) 122188, CBS 251.72, CBS 143.77, CBS 272.77, CBS122190, CBS122189, and VKM F-3500D. Exemplary C1 derivatives include modified organisms where one or more endogenous genes or sequences have been deleted or modified and / or one or more heterologous genes or sequences have been introduced. Derivatives include, but are not limited to, UV18 # 100f Δalpl, UV18 # 100f Δpyr5 Δalp1, UV18 # 100.f Δalp1 Δpep4 Δalp2, UV18 # 100.f Δpyr5 Δalp1 Δpep4 Δalp2 and UV18 # 100.f Δpyrp Δppp2 , as described in WO 2008073914 and WO 2010107303.

In some embodiments, the host cell may be of the Trichoderma species, such as T. longibrachiatum, T. viride, Hypocrea jecorina or T. reesei, T. koningii, and T. Harzianum Alternatively, the host cell may be of the species, Aspergillus, such as A. awamori, A. funigatus, A. japonicus, A. nidulans, A. niger, A. aculeatus, A. foetidus, A. oryzae, A. sojae, and A. kawachi. Alternatively, the host cell is of the Fusarium species, such as F. bactridioides, F. cerealis, F. crookwellense,

F. culmorum, F. graminearum, F. graminum. F. oxysporum, F. roseum, and F. venenatum.

The host cell can also be of the Neurospora species, such as N. crassa. Alternatively, the host cell is of the Humicola species, such as H. insolens, H. grisea, and H. lanuginosa. Alternatively, the host cell is of the Mucor species, such as M. miehei and M. circinelloides. The host cell can be of the Rhizopus species, such as R. oryzae and R. niveus. Alternatively, the host cell is of the Penicillum species, such as P. purpurogenum, P. chrysogenum, and P. verruculosum.

Alternatively, the host cell is of the Thielavia species, such as T. terrestrial. Alternatively, the host cell is of the Tolypocladium species, such as T. inflatum and T. geodes. Alternatively, the host cell is of the Trametes species, such as T. villosa and T. versicolor Alternatively, the host cell is of the Chrysosporium species, such as C. lucknowense, C. keratinophilum, C. tropicum, C. merdarium, C. inops, C. pannicola, and C. zonatum. In a particular embodiment, the host is C. lucknowense.

Alternatively, the host cell is an algae like Chlamydomonas (like C. reinhardtii) and Phormidium

(P. sp. ATCC29409).

Alternatively, the host cell is a prokaryotic cell. Suitable prokaryotic cells include Gram-positive, Gram-negative and Gram-variable bacterial cells. Examples of bacterial host cells include Bacillus species (such as B. subtilis, B. licheniformis, B. megaterium, B. stearothermophilus and

B. amyloliquefaciens), Streptomyces (for example, S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, and S. lividans), and Streptococcus (as S. equisimiles, S. pyogenes, and S. uberis).

Non-limiting examples of cell types in this section include Aspergillus aculeatus, Azospirillum irakense KBC1, Bacillus sp. GL1, Cellulomonas biazotea, Clostridium thermocellum, Thermoanaerobacter brockii, Coccidioides posadasii, Dictyostelium discoideum, Elizabethkingia meningoseptica, Erwinia chrysanthemi, Escherichia coli, Gluconacetobacter xylinus, Hypocrea jecorinata, Kuraishia capsule, Kuraicia capsule, Kum. C7, Penicillium brasilianum, Periconia sp. BCC 2871, Phaeosphaeria avenaria, Prevotella albensis, Rhizobium leguminosarum, Rhizomucor miehei, Ruminococcus albus, Saccharomycopsis fibuligera, Salmonella

typhimurium, Septoria lycopersici, Streptomyces coelicolor, Talaromyces emersonii, Thermotoga maritima, Tropaeolum majus, Uromyces viciae-fabae, and Wickerhamomyces anomalus.

Strains that can be used in the practice of the invention (both prokaryotic and eukaryotic strains) can be obtained from any suitable source, including, but not limited to, the American Type Culture Collection (ATCC) or other biological deposits such as Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and the Collection of Patent Crops for Agricultural Research Service, Northern Regional Research Center (NRRL).

Host cells can be genetically modified to have characteristics that improve genetic manipulation, protein secretion, protein stability or other desirable properties for protein expression or secretion. For example, blocking the Alp1 function results in a cell that is protease deficient. Blocking the pyr5 function results in a cell with a pyrimidine deficient phenotype. Host cells can be modified to eliminate endogenous cellulase protein coding sequences or otherwise eliminate the expression of one or more endogenous cellulases. The expression of one or more unwanted endogenous cellulases can be inhibited to increase the proportion of cellulases of interest, for example, by chemical or UV mutagenesis and subsequent selection. Homologous recombination can be used to induce gene-directed modifications by specifically targeting a gene in vivo to suppress the expression of the encoded protein.

Transformation and Cell Culture

The polynucleotides of the disclosure, which encode GH61 proteins, cellulase proteins or other proteins, can be introduced into host cells for expression. The polynucleotide can be introduced into the cells as a self-replicating episome (eg, expression vector), or it can be stably integrated into the host cell's DNA.

The introduction of a vector or a DNA construct into a host cell can be carried out by any suitable method, including but not limited to, calcium phosphate transfection, DEAE-Dextran-mediated transfection, electroporation or other common techniques (See Davis et al. ., 1986, BASIC METHODS IN MOLECULAR ENBIOLOGY; Sambrook et al (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, New York; "Guide to Yeast Genetics and Molecular Biology", C. Guthrie Y

G. Fink, Eds., Methods in Enzymology 350 (Academic Press, San Diego, 2002). In some embodiments, the polynucleotide that is introduced into the host cell remains in the genome or in the plasmid or other vector stably maintained in the cell and is capable of inheriting from the progeny. Stable transformation is typically performed by transforming the host cell with an expression vector comprising the polynucleotide of interest junco with a selectable marker gene (for example, a gene that confers resistance to an antibiotic). Only those host cells that have integrated polynucleotide sequences of the expression vector into their genome will survive selection with the marker (eg, antibiotic). These stably formed host cells can then be propagated according to methods known in the art.

The constructed host cells can be cultured in modified conventional nutrient media as appropriate to activate promoters, select transformants or amplify the GH61 polynucleotide. General references in cell culture and nutrient media techniques include GENETIC MANIPULATION IN FUNGI, Benett, J. W., et al., Ed. Academic Press, 1985; MORE GENETIC HANDLING IN FUNGI, Benett, J. W., et al., Ed. Academic Press, 1991; AND THE MICROBIOLOGICAL MEDIA MANUAL, CRC Press, Boca Raton, FL, 1993. Culture conditions for C1 host cells are described in US 2008/0194005, US 2003/0187243, WO 2008/073914 and WO 01/79507. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art. As noted, many references are available that describe the culture and production of many cells, including cells of bacteria, plants and animals (especially mammals) and archaea. Atlas and Parks (eds.). The Microbiological Media Manual (1993) CRC Press, Boca Ratón, FL. More information for cell culture is found in commercial literature available as a Cell Culture Catalog for Living Science Research (1998) from Sigma-Aldrich, Inc (St. Louis, MO) ("Sigma-LSRCCC") and, for example, The Catalog of Plant Crops and Complements (1997) also by Sigma-Aldrich, Inc (St. Louis, MO) ("Sigma-PCCS").

Protein Enrichment and Purification

An expressed polypeptide can be recovered from cells or broth. Optionally, a protein can be enriched (for example, purified or partially purified) using methods well known in the art. For example, the polypeptide can be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, chromatography (e.g., ion exchange chromatography, solid phase bond, affinity, interaction hydrophobic, chromato focus and size exclusion) and / or filtration or precipitation. The stages of refolding the protein can

be used, as desired, to finalize the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be used in the final stages of purification. See, for example, Parry et al., 2001, Biochem. J. 353: 117, and Hong et al., 2007, Appl. Microbiol Biotechnol 73: 1331. Other purification methods well known in the art include those set forth in Sandana (1997) Protein Bioseparation, Academic Press, Inc .; Bollag et al. (1996) Protein Methods, 2nd Edition, Wiley-Liss, NY; Walker (1996) The Protein Protocol Manual, Humana Press, NJ; Harris and Angal (1990) Protein Purification Applications: A Practical Technique, IRL Press in Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice, 3rd Edition, Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: High Resolution Principles, Methods and Applications, Second Edition, Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM, Humana Press, NJ; PURIFICATION OF PROETINS: PRINCIPLES, HIGH RESOLUTION METHODS AND APPLICATIONS, J. C. Janson (Ed.), Wiley 2011; EXPRESSION AND PURIFICATION OF SURVEILLANCE PROTEINS: METHODS AND PROTOCOLS, S.A., Doyle (Ed.) Humana Press 2009.

General techniques

Polynucleotides encoding GH61 proteins and other proteins can be prepared, for example, by chemical synthesis using the classical phosphoramidite method described in Beaucage, et al., 1981, Tetrahedron Letters, 22: 1859-69 or the method described by Matthes, et al., 1984, EMBO J. 3: 801-05. Oligonucleotides of up to about 40 bases are synthesized individually, then bound (for example, by enzymatic or chemical ligation methods, or polymerase mediated methods) to form essentially any desired continuous sequence.

General texts describing molecular biological techniques that include the use of vectors, promoters, in vitro amplification methods that include polymerase chain reaction (PCR) and ligase chain reaction (CSF) are Berger and Kimmel, GUIDE TO MOLECULAR CLONING TECHNIQUES, ENZYMOLOGY METHODS volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., MOLECULAR CLONING - A LABORATORY MANUAL (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, FM Ausubel et al., eds. Current Protocols (as a supplement throughout 2009).

VII. Purification of endogenous GH61 proteins from culture broth

As an alternative to the recombinant expression of HG61 proteins of this disclosure, secreted GH61 proteins can be fractionated from the culture broth of Myceliophthora thermophila that produces and secretes a

or more endogenous proteins with GH61 activity. In the same way, endogenous GH61 not secreted can be recovered by lysis of M. thermophila cells.

The GH61 proteins of this disclosure can be obtained from cells expressing GH61 proteins using standard protein separation techniques, such as those described above, and following GH61 activity during fractionation with a suitable GH61 activity assay.

As illustrated in the Examples, when the M. thermophila culture broth protein is isolated, an effective combination is chromatography on a phenyl group having resin, followed by anion exchange chromatography. As a result of the separation techniques, the specific activity of the GH61 protein (the activity observed in an activity test per total unit of protein) may increase by about 10, about 25, about 100, about 250, about 1000 times or more.

Once the GH61 activity has been fractionated from a suitable source, the fractions can be recombined with each other and / or with recombinant GH61 proteins in any combination (see Examples). Such fractions or combinations can then be used to promote the activity of one or more cellulases, as described herein. The purified or recombinant GH61 proteins of this disclosure, and combinations thereof, may cause an increase in the cellulase activity index for conversion of cellulosic biomass or other substrate to fermentable sugars by at least about 1%, 5%, 10%, 15%, 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about doubling, at least about quadrupling or more.

When using such protein separation techniques in combination with a GH61 activity assay, it has been determined that protein fractions having complete or partial sequence data corresponding to GH61f, GH61a, GH61v, GH61p, GH61g, and GH61i (SEQ ID NOs: 2, 7, 13, 20, 21, 23, 26) have the ability to improve cellulase activity according to their classification as a GH61 protein (See Examples).

VIII: Cellulases

The GH61 proteins of this disclosure are useful for increasing the production of fermentable sugars in a saccharification reaction with one or more cellulase enzymes. The GH61 protein and cellulase enzymes can be produced in the same cell or in different cells. In each case, cellulase enzymes can be expressed from a recombinant coding region or from a constitutive gene. Cellulase enzymes can be provided in the form of a culture broth or supernatant, or purified to the extent desired.

Cellulases for use in the present invention may be derived from any organism that produces cellulases, and may be expressed in, for illustration but without limitation, any cell described herein. In some embodiments, cellulases are derived from and / or expressed in a filamentous fungus (for example, Myceliophthora species, Aspergillus Azospirillum, and Trichoderma) or yeast cell. For illustration, cellulase derived from any of the following cells can be used. For example, many fungi (including, but not limited to, species of Thielavia, Humicola, Chaetomium, Neurospora, Chaetomidium, Botryosphaeria, Trichophaea, Aspergillus, Schizophyllum, Agaricus, Sporotrichium, Corynascus, Myceliophthora, Acremonium, Thermoaschaeternia, Phyloacterium, Clamo, Botany , Cochliobolus, Cryphonectria, Emericella, Fusarium, Gibberella, Hypocrea, Irpex, Magnaporthe, Nectria, Neosartorya, Penicillium, Phanerochaete, Pleurotus, Podospora, Polyporus, Sclerotinia, Sordaria, Talaromyces, Trichodermonium, and Volvarieusus, example Volvarieus, ; Alternaria alternate; Aspergillus aculeatus; Aspergillus clavatus; Aspergillus flavus; Aspergillus fumigatus; Aspergillus nidulans; Aspergillus niger; Aspergillus oryzae; Aspergillus terreus; Botryotinia fuckeliana; Chetomiumulaumiumumumumumumumumumumumum; oxysporum; Fusarium poae; Fusarium venenatum; Gibberella avenacea; Gibberella pulicaris; Gibberella zeae; Humisala grisea; Hypocrea koningii; Hypocrea lixii; Hypocrea virens; Irpex lacteus; Magnaporthe grisea; Nectria haematococca; Neosartorya fischeri; Neurospora crassa; Penicillium chrysogenum; Penicillium decumbens; Penicillium funiculosum; Penicillium janthinellum; Penicillium marneffei; Penicillium occitanis; Penicillium oxalicum; Phanerochaete chrysosporium; Pleurotus sp. 'Florida'; Podospora anserine; Polyporus arcularius; Sclerotinia sclerotiorum; Sordaria macrospora; Talaromyces emersonii; Talaromyces stipitatus; Thermoascus aurantiacus; Trichoderma sp .; Trichoderma viride; Trichoderma reseipdb; Volvariella volvacea. In one embodiment the cell is M. thermophila.

Endoglucanase (EG)

The disclosure provides a cell that expresses a GH61 protein in combination with a recombinant endoglucanase. The terms "endoglucanase" or "EG" refer to a group of cellulase enzymes classified as E.C. 3.2.1.4. These enzymes catalyze the hydrolysis of internal β-1,4 glycosidic bonds.

For example, the cell may contain a recombinant polynucleotide sequence encoding the EG protein. The EG polynucleotide sequence may be operably linked to a heterologous promoter and / or the EG polypeptide sequence comprises a sequence signal. The EG protein can be expressed as a preprotein, which is secreted from the cell with the simultaneous loss of the signal peptide.

EG may comprise an endogenous endoglucanase of M. thermophila as EG2a of M. thermophila (see WO 2007/109441) or a variant thereof. EG may be S. avermitilis, with a sequence set forth in GenBank access NP_821730, or a variant as described in US 2010/0267089 A1. EG can be a Thermoascus aurantiacus EG, or an endogenous EG of a bacterium, a yeast, or a filamentous fungus different from M. thermophila. In fact, it is contemplated that any suitable EG will find use in combination with the GH61 proteins provided herein. It is not intended that the present invention be limited to any specific EG.

β-Glucosidase (BGL)

The disclosure provides a cell that expresses a GH61 protein in combination with a recombinant β-glucosidase. The terms "β-glucosidase", "cellobiodease" or "BGL" refer to a group of cellulase enzymes classified as E.C. 3.2.1.21. These enzymes hydrolyze cellobiose to glucose.

For example, the cell may contain a recombinant polynucleotide sequence encoding the BGL protein, where the polynucleotide sequence is operably linked to a heterologous promoter and / or signal sequence. The BGL protein can be expressed as a pre-protein, which is secreted from the cell with simultaneous loss of the signal peptide.

BGL can be an M. thermophila BGL1 or a variant thereof. The BGL1 may comprise the sequence set forth in SEQ ID NO: 60 or SEQ ID NO: 66, or is a variant thereof, or a variant described in US 2011/0129881 A1. Alternatively, the BGL is from Thermoascus aurantiacus (TaBGL), which has a sequence set forth as SEQ ID NO: 61, or is a variant thereof, or a variant such as those described in US 2011/0124058 A1.

Alternatively, the BGL is from Azospirillum irakense (CelA), which has a sequence set forth as SEQ ID NO: 62, or a variant thereof, or a variant described in US 2011/0114744 A1. Alternatively, BGL is described in TABLE 1 of PCT application No. PCT / US2010 / 038902. Alternatively, BGL is an endogenous BGL of a bacterium, a yeast, a filamentous fungus different from M. thermophila. The use of naturally occurring variants of BGLs is also contemplated. In fact, it is contemplated that any suitable BGL will find use in combination with the GH61 proteins provided herein. It is not intended that the present invention be limited to any specific BGL.

Type 1 and Type 2 Cellobiohydrolase

The disclosure provides a cell that expresses a GH61 protein in combination with a recombinant type 1 cellobiodrilase. The terms "cellobiohydrolase," "exoglucanase," exo-cellobiodrilase "or" CBH "refer to a group of cellulase enzymes classified as E.C. 3.2.1.91. Type 1 cellobiohydroases (CBH2) hydrolyse cellobiose in a process of the reducing end of the cellulose chains. Type 2 cellobiodrilasas (CBH3) hydrolyse cellobiose in a non-reducing process of cellulose chains.

For example, the cell may contain a recombinant polynucleotide sequence encoding the CBH protein, where the polynucleotide sequence is operably linked to a heterologous promoter and / or signal peptide sequence. The CBH protein can be expressed as a pre-protein, which is secreted from the cell with simultaneous loss of the signal peptide.

The cell can be an M. thermophila cell, and it can be an endogenous celloiodiodollase, such as CBH1a, which has a sequence set forth in SEQ ID No. 63 or 67 or a variant thereof. Alternatively, CBH1 is an endogenous CBH1 of a bacterium, a yeast, a filamentous fungus other than M. thermophila or a naturally occurring variant of CBH1s. In fact, it is contemplated that any suitable CBH will find use in combination with the GH61 proteins provided herein. It is not intended that the present invention be limited to any specific CBH.

IX .: Cell-free compositions where GH61 protein is combined with cellulase enzymes

In one aspect, the disclosure provides a composition comprising at least one GH61 protein described herein (for example, comprising a sequence of SEQ ID NO. 1-30, comprising a secreted part of the GH61 protein, comprising a truncated part terminal amino acid of the GH61 protein, and biologically active variants thereof), in combination with at least one, at least two, at least three or more cellulases selected from EGs, BGLs, CBH1s and / or CBH2s, where the combined mass of EG, BGL, CBH1 and / or CBH2 is at least about 50%, at least about 60% or at least about 70% of the total cell-free protein in the composition. The GH61 protein (either in broth or in partially purified form) can be combined with M. thermophila cellulases or other cellulase producing organisms (including, for example, organisms listed below).

In some compositions of the invention, the GH61 protein comprises SEQ ID NO: 2; and (a) CBH1 is a CBH1a variant of M. thermophila with at least about 80%, at least about 85%, sometimes at least about 90%, and sometimes at least about 95% sequence identity with SEQ ID NO: 63 or 67; and / or (b) CBH2 is a CBH2b variant of M. thermophila with at least about 80%, at least about 85%, sometimes at least about 90%, and sometimes at least about 95% sequence identity with SEQ ID NO: 64 or 68; and / or (c) the BGL is a BGL1 variant of M. thermophila with at least about 80%, at least about 85%, sometimes at least about 90%, and sometimes at least about 95% sequence identity with SEQ ID NO: 60 or 66.

The composition may also be a cell culture medium (that is, culture broth) containing recombinant secreted GH61 and cellulase proteins. Tale media can be produced by culturing recombinant cells described hereinbefore under conditions where a combination of enzymes (e.g., GH61, EG, CBH and / or BGL proteins) is expressed and secreted. The cell culture medium can be essentially free of cells, for example, removing them by centrifugation or filtration. A composition for degrading cellulose can be produced by culturing recombinant cells described above under conditions where enzymes (for example, GH61, EG, CBH and / or BGL proteins) are expressed and secreted, optionally removing cells from the medium, and optionally enriching the medium for Increase protein concentration.

X. Use of GH61 proteins in saccharification reactions

The saccharification reactions can be performed by exposing a cellulosic substrate (eg, pretreated biomass) to a GH61 protein and cellulase, which work in concert to hydrolyze a cellulose and produce fermentable sugars.

Typically, cellulases include at least one endoglucanase (EG), at least one β-glucosidase (BGL), at least one Type 1 cellobiohydrolase (CBH1), and / or at least one Type 2 cellobiohydrolase (CBH2).

The cells and compositions of the invention (including the culture broth or cell lysates) can be used in the production of fermentable sugars from cellulosic biomass. The biomass substrate can be converted to a fermentable sugar (a) optionally pre-treating a cellulosic substrate to increase its susceptibility to hydrolysis; (b) contacting the optionally pretreated cellulosic substrate of step (a) with a composition, culture medium or cell lysate containing a GH61 protein and cellulases under conditions suitable for the production of cellobiose and fermentable sugars (eg glucose ).

In one embodiment, to perform a saccharification reaction, each of the GH61 proteins and cellulase enzymes referred to above can be partially or substantially purified, and the purified proteins are combined with the cellulosic substrate. In another embodiment the various individual proteins are recombinantly expressed in different cells, and the media containing the secreted proteins are added to the biomass.

The compositions may react with the substrate at a temperature in the range of about 25 ° C to about 110 ° C, from about 30 ° C to about 90 ° C, from about 30 ° C to about 80 ° C, from about 40 ° C to about 80 ° C, of approximately 35 ° C to approximately 75 ° C, from approximately 55 ° C to approximately 100 ° C or approximately 90 ° C. The process can be carried out at a pH in a range of from about pH 3.0 to about 8.5, from about pH 3.5 to about 8.5, from about pH 4.0 to about 7.5, from about pH 4.0 to about 7.0 and from about pH 4.0 to about 6.5. The reaction times to convert a particular biomass substrate into a fermentable sugar can vary but the optimal reaction time can be easily determined. Exemplary reaction times may be in the range of from about 1 to about 240 hours, from about 5 to about 180 hours and from about 10 to about 150 hours. For example, the incubation time may be at least 1 hour, at least 5 hours, at least 10 hours, at least 15 hours, at least 25 hours, at least 50 hours, at least 100 hours or at least 180 hours.

In some embodiments, the GH61 polypeptides of the present disclosure are used in combination with other optional ingredients such as at least one buffer or a surfactant. In some embodiments, at least one buffer with the GH61 polypeptide of the present disclosure (optionally combined with other enzymes) is used to maintain a desired pH in the solution where GH61 is used. Suitable buffers are well known in the art. In some embodiments, at least one surfactant with GH61 of the present disclosure is used. Cations (for example, Cu ++, Mn ++, Co ++, Mg ++ and Ca ++ in concentrations of 0.001 to 50 mM, 5 µM to 1 mM, 10-50 µM to 10-20 µM) can be included in the reaction.

Exemplary combinations of the GH61 protein and cellulases include: GH61 protein with one or more endoglucanases (EG); (BGL); GH61 protein with one or more 1 (CBH1); or GH61 protein with one or more Type 2 cellobiohydrolases (CBH2). Other combinations are GH61 protein with EG and BGL; GH61 protein with EG and CBH1; GH61 protein with EG and CBH2; GH61 protein with BGL and CBH1; GH61 protein with BGL and CBH2, or GH61 protein with CBH1 and CBH2. Other combinations are GH61 protein with EG, BGL, and CBH1; GH61 protein with EG, BGL, and CBH2; GH61 protein with EG, CBH1, CBH2; GH61 protein with BGL, CBH1, and CBH2; and GH61 protein with all of EG, BGL, CBH1, and CBH2. Other enzymes listed in this disclosure may be included in one or more of these combinations.

In some embodiments of the disclosure, the enzyme mixture comprises an isolated GH61 as provided herein and at least one or more of a type 1 celloiodiodollase such as CBH1a, an isolated CBH2b, an isolated endoglucanase (EG) such as a type 2 endoglucanase (EG2), or a type 2 endoglucanase (EG2), and / or an isolated β-glucosidase (BGL). In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at minus 50% of the enzyme mixture is GH61. In some embodiments, the above mixture further comprises a type 1a celloiodiodollase (e.g., CBH2a), and GH61, where the enzymes together comprise at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the enzyme mixture. In some embodiments, the enzyme mixture further comprises a β-glucosidase (BGL), GH61, CBH2b, where the three enzymes together comprise at least 30%, at least 35%, at least 40%, at least 45%, at minus 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or at least 85% of the enzyme mixture. In some embodiments, the enzyme mixture further comprises an endoglucanase (EG), GH61, CBH2b, CBH1a, BGL where the five enzymes together comprise at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% of the enzyme mixture. In some embodiments, the enzyme mixture comprises GH61, CBH2b, CBH1a, BGL and at least one EG, in any proportion suitable for the desired reaction.

In some embodiments, the composition of the enzyme mixture comprises cellulases isolated in the following proportions by weight (where the total weight of the cellulases is 100%); approximately 20% -10% BGL, approximately 30% -25% CBH1a, approximately 10% -30% GH61, approximately 20% -10% EG1b, and approximately 20% -25% CBH2b. In some embodiments, the enzyme mixture comprises isolated cellulases in the following proportions by weight: approximately 20% -10% GH61, approximately 25% -15% BGL, approximately 20% -30% CBH1a, approximately 10% -15 % of EG, and about 25% -30% of CBH2b. In some embodiments, the enzyme mixture comprises isolated cellulases in the following proportions by weight: approximately 30% -20% GH61, approximately 15% -10% BGL, approximately 25% -10% CBH1a, approximately 25% -10 % CBH2b, approximately 15% -10% EG. In some embodiments, the enzyme mixture comprises isolated cellulases in the following proportions by weight: approximately 40-30% GH61, approximately 15% -10% BGL, approximately 20% -10% CBH1a, approximately 20% -10% of CBH2b, and about 15% -10% of EG.

In some embodiments, the enzyme mixture comprises isolated cellulases in the following proportions by weight: approximately 50-40% GH61, approximately 15% -10% BGL, approximately 20% -10% CBH1a, approximately 15% -10% of CBH2b, and about 10% -5% of EG. In some embodiments, the enzyme mixture comprises isolated cellulases in the following proportions by weight: approximately 10% -15% GH61, approximately 20% -25% BGL, approximately 30% -20% CBH1a, approximately 15% -5 % of EG, and about 25% -35% of CBH2b. In some embodiments, the enzyme mixture comprises isolated cellulases in the following proportions by weight: approximately 15% 5% GH61, approximately 15% -10% BGL, approximately 45% -30% CBH1a, approximately 25% -5% of EG1b, and about 40% -10% CBH2b. In some embodiments, the enzyme mixture comprises isolated cellulases in the following proportions by weight: approximately 10% GH61, approximately 15% BGL, approximately 40% CBH1a, approximately 25% EG, and approximately 10% CBH2b.

In some embodiments, the enzyme component comprises more than one CBH2b, CBH1a, EG, BGL and / or GH61 enzyme (eg, 2, 3 or 4 different enzymes) in any suitable combination. In some embodiments, an enzyme blend composition of the invention further comprises at least one additional protein and / or enzyme. In some embodiments, the enzyme blend compositions of the present invention further comprise at least one additional enzyme other than GH61, BGL, CBH1a, GH61 and / or CBH. In some embodiments, the enzyme blend compositions of the invention further comprise an additional cellulase, different from the variant of GH61, BGL, CBH1a, GH61 and / or CBH. In some embodiments, the GH61 polypeptide of the invention is also present in mixtures with non-cellulase enzymes that degrade cellulose, hemicellulose, pectin and / or lignocellulose and / or enzymes described herein below.

Exemplary embodiments of M. thermophila

For illustration and not limitation, the following exemplary embodiments are provided:

An embodiment of the disclosure is a host cell that is a M. tehrmophila cell that expresses a recombinant protein comprising SEQ ID NO: 1-30, which comprises a secreted part of the GH61 protein, which comprises a truncated amino terminal portion of GH61 protein and biologically active variants thereof. In some cases, the cell expresses a GH61 selected from SEQ ID Nos: 3 to 12 or the corresponding secreted protein, or a GH61 selected from SEQ ID Nos: 13 to 256 or the corresponding secreted protein.

The disclosure provides a cell comprising a recombinant nucleic acid sequence encoding a GH61 protein. In some aspects, the disclosure provides a cell comprising a recombinant nucleic acid sequence encoding a protein with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90 %, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with SEQ ID NO: 2, or at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least approximately 98% or at least about 99% sequence identity with the secreted part of any one of SEQ ID Nos: 1 to 30, comprising a secreted part of the GH61 protein (eg, SEQ ID NO: 2), which comprise a truncated amino terminal portion of the GH61 protein, and biologically active variants thereof. The recombinant nucleic acid sequence can encode a protein with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least

about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with a GH61 protein listed in TABLE 1 or TABLE 2.

The nucleic acid may comprise the nucleotide sequence shown in any of SEQ ID Nos: 31 to 59, or a fragment thereof, or a nucleic acid that hybridizes with SEQ ID Nos: 31-59 (or the exactly complementary sequence) under severe conditions (that is, high, high or very high average severity), and that encodes a polypeptide with GH61 activity. Alternatively, the nucleic acid may encode a polynucleotide that is at least about 70%, at least about 80%, at least about 90% or at least about 95% or at least about 99%, identical to any such sequence or fragment, where the nucleic acid encodes and can be expressed to provide a polypeptide with GH61 activity. Optionally, such nucleic acid sequences can be optimized with codon for expression in a particular species, such as a yeast, as described elsewhere in this disclosure.

In one embodiment of the disclosure, a host cell expresses at least one recombinant GH61 comprising any one of SEQ ID Nos: 1 to 30, and / or at least one recombinant GH61 protein having at least about 70%, at least about 75 %, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with SEQ ID Nos: 1 to 25; and also expresses:

a) a recombinant EG protein with at least about 70%, at least about 75%, at least about 80%, at least about 85%, sometimes at least about 90% and sometimes at least about 95% sequence identity with M. thermophila EG2a (SEQ ID NO: 65); I

b) a recombinant CBH1a protein with at least about 70%, at least about 75%, at least about 80%, at least about 85%, sometimes at least about 90% and sometimes at least about 95% sequence identity with SEQ ID NO: 63 or 67; I

c) a recombinant CBH2b protein with at least about 70%, at least about 75%, at least about 80%, at least about 85%, sometimes at least about 90% and sometimes at least about 95% sequence identity with SEQ ID NO: 64 or 68; I

d) a recombinant BGL protein with at least about 70%, at least about 75%, at least about 80%, at least about 85%, sometimes at least about 90% and sometimes at least about 95% sequence identity with SEQ ID NO: 60, 61, 62 or 66.

In certain embodiments of the disclosure, the cell expresses at least one, at least two, at least three (eg, b-d) or all four of (a), (b), (c) and (d).

XI Saccharification in the absence of exogenous EG

In one aspect, the disclosure provides a method for hydrolyzing a cellulosic substrate comprising combining a GH61 protein with enzymes of β-glucosidase (BGL) and celloiodiodollase (CBH), in a composition substantially free of endoglucanase (EG). It will be appreciated that the EG type activity to which a GH61 protein contributed is not considered an endoglucanase. As illustrated in the example below, a saccharification reaction can be performed in the presence of a GH61 protein, a β-glucosidase, and one or more celloiodiodollase enzymes, without recombinant EG or without added EG, or substantially free of EG. Type 1 celloiodiodollase

or Type 2 or both may be present. In the absence of EG, GH61 can increase the production of a simple BGL and CBH saccharification reaction by more than 1.5 or 1.7 times.

A reaction is said to be "substantially free" of endoglucanase if (a) there is no detectable endoglucanase activity in the reaction, or (b) the amount of that EG enzyme present is less than 2%, often less than 1%, often less than 0.5%, often less than 0.2% and often less than 0.1% (w / w) of the amount of BGL present, or (c) the amount of that EG enzyme present is less at 2%, often less than 1%, often less than 0.5%, often less than 0.2% and often less than 0.1% (w / w) of the amount of CBH present, or ( d) the amount of that EG enzyme present is less than 2%, often less than 1%, often less than 0.5%, often less than 0.2% and often less than 0.1% (w / p) of the amount of GH61 present, or (e) the amount of that EG enzyme present is less than 2%, often less than 1%, often less than 0.5%, often less than 0.2% and often less than 0.1% (w / w) of the total amount of cellulose present.

XII. Compositions comprising other enzymes

Additional enzymes that can act in concert to hydrolyze a cellulosic substrate (such as cellulose or a starch-containing substrate) in the saccharification process can be included in the compositions.

or incorporated into the methods of this invention. Such enzymes include, but are not limited to, xylanases, hemicellulases, amylases, esterases and cellulases, α-glucosidases, aminopeptidases, carbohydrases, carboxypeptidases, catalase, chitinases, cutinases, cyclodextrin glycosyltransferases, deoxyribonucleases, αgagagacosases, αgagagarases, αgagagarases, αgalucosidases, αgalucosases , invertases, lacases, lipases, mannosidases, mutanases, oxidases, pectinolytic enzymes, peroxidases, phospholipases, phytases, polyphenoloxidases, ribonucleases and trans-glutaminases, as well as other celluloses (for example, type 1 and type 2 celloiodiodlases), endoglucanases . Cellulase mixtures for efficient enzymatic hydrolysis of cellulose are well known (See, for example, Viikari et al., 2007, Adv. Biochem. Eng. Biotechnol., 108: 121-45; and United States patent publications 2009 / 0061484; US 2008/0057541; and US 2009/0209009). In some embodiments, mixtures of purified enzymes that occur naturally or recombinantly are combined with cellulosic feedstock or a cellulose hydrolysis product. In some embodiments, one or more populations, each producing one or more naturally occurring or recombinant celluloses, are combined with cellulosic raw material or a cellulose hydrolysis product.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one endoxylanase. Endoxylanases (EC 3.2.1.8) catalyze the endohydrolysis of 1,4-β-D-xyloid bonds in xylanes. This enzyme can also be referred to as endo-1,4-β-D-xylanase or 1,4-β-D-xylan xylanhydrolase. In some embodiments, an alternative is EC 3.2.1.136, or glucuronoarabinoxylan endoxylanase, an enzyme that is capable of hydrolyzing 2.4 xyloid bonds in glucuronoarabinoxylanes.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one β-xylosidase. β-xylosidases (EC 3.2.1.37) catalyze the hydrolysis of 1,4-β-D-xylanes, to remove successive D-xylose residues from the non-reducing terminals. This enzyme may also be referred to as xylan, 1,4-β-xylosidase, 1,4-β-D-xylan xylohydroase, exo 1,4-β-xylosides or xylobiase.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one α-L-arabinofuranosidase. α-L-arabinofuranosidases (EC 3.2.1.55) catalyze the hydrolysis of non-terminal alpha-Larabinofuranosidase residues. The enzyme acts on alpha-L-arabinofuranosidases, alpha-L-arabinans containing (1,3) -and / or (1,5) -unions, arabinoxylans and arabinogalactans. Alpha-L-arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-Llarabinosidase, alpha-arabinofuranosidase, arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidase hydrolase, Larabinosidaan and alpha-L-arabinan

In some additional embodiments, the present disclosure provides at least one GH61 and at least one alpha-glucuronidase. Alpha-glucuronidase (EC 3.2.1.139) catalyzes the hydrolysis of an alpha-D-glucuronidase to Dglucuronate and an alcohol.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one acetyloxylanesterase. Acetyloxylanesterases (EC 3.1.72) catalyze the hydrolysis of acetyl group of polymeric xylan, acetylated xylose, acetylated glucose, alpha-naphthyl acetate, and p-nitrophenyl acetate.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one feruloyl esterase. Feruyl esterases (EC 3.1.1.73) have a 4-hydroxy-3-methoxycinnamoyl sugar hirolase activity (EC 3.1.1.73) that catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar, which it is normally arabinose in "natural" substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I or FAE-II.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one coumaroyl esterase. Coumaroil esterases (EC 3.1.1.73) catalyze a reaction of the form: coumaroil-saccharide + H2O) coumarate + saccharide. In some embodiments, the saccharide is an oligosaccharide or a polysaccharide. This enzyme can also be referred to as trans-4-coumaroil esterase, trans-p-coumaroil esterase, p-coumaroil esterase or p-coumaric acid esterase. The enzyme is also classified in EC 3.1.1.73 so that it can also be referred to as feruoyl esterase.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one alpha-galactosidase. Alpha-galactosidates (EC 3.2.1.222) catalyze the hydrolysis of non-terminal β-D-galactose residues in β-D-galactosides. In some embodiments, the polypeptide is also capable of hydrolyzing αL-arabinosides. This enzyme can also be referred to as exo- (1-gt; 4) -β-D-galactanase or lactase.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one beta-mannanase. Beta-mannanases (EC 3.2.1.78) catalyze the random hydrolysis of manidic 1,4-β-D bonds in mannan, galactomannan and glucomannan. This enzyme can also be referred to as mannan endo-1,4-β-manidase or endo1,4-mannanase.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one beta-mannosidase. Beta-mannosed (EC 3.2.1.25) catalyze the hydrolysis of non-terminal β-D-mannose residues in β-D-mannosides. This enzyme can also be referred to as mananansa or manasa.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one glucoamylase. Glucoamylases (EC 3.2.1.3) catalyze the release of D-glucose from non-reducing ends of oligo-and poly-saccharide molecules. Glucoamylase is often considered a type of amylase known as amyl glucosidase.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one amylase. Amylases (EC 3.2.1.1) are starch-dividing enzymes that degrade starch and related compounds by hydrolyzing the α-1,4 and / or α-1-6 glycosidic bonds in a way that acts endo-or exo-. Amylases include α-amylases (EC 3.2.1.1); β-amylases (3.2.1.2), amylo-amylases (EC 3.2.1.3), α-glucosidases (EC 3.2.1.20), pululanases (EC 3.2.1.41) and isoamylases (EC 3.2.1.68). In some embodiments, the amylase is α-amylase. In some embodiments one or more enzymes that degrade pectin are included in the enzyme mixtures comprising GH61 of the present disclosure. A pectinase catalyzes the hydrolysis of pectin into smaller units such as oligosaccharide or monomeric saccharides. In some embodiments, the enzyme mixtures comprise any pectinase, for example an edo-polygalacturonase, a pectin methyl esterase, an endogalactanase, an acetyl esterase pectin, ujna endo-pectin lyase, lycta pectate, alpha ramosidase, exo-galacturonase, an exo polygalacturoanto lyase, a ramnogalacturonano hirolasa, a ramnogalacturonano liasa, a ramnogalacturonano acetyl esterase, a ramnogalacturonano galacturonohydrolase and / or a xylogalactruronase.

In some embodiments, the present disclosure provides at least one GH61 and at least one endopolygalacturonase. Endo-polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1,4, α-Dgalactosiduronic bonds in pectate and other galacturonanos. This enzyme can also be referred to as polygalacturonase pectin depolymerase, pectinase, endopolygalacturonase, pectinase, pectin hydrolase, pectin polygalacturonase, poly-α-1,4-galacturonide glycanhydrolase, endogalacturonase; endo-D-galacturonase or poly (1,4-α-D-galacturonide) glycanhydrolase.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one methyl pectin mats. Pectin methyl esterases (EC 3.1.1.11) catalyze the reaction: pectin + n H2O = n methanol + pectate. The enzyme can also be known as pectinesterase, pectin, demethoxylase, pectin methoxylase, pectin methylesterase, pectase, pectinaserase or pectin pectylhydrolase.

In some additional embodiments, the present disclosure provides at least one endo-galactanase. Endo-galactanases (EC 3.2.1.89) catalyze the endohydrolysis of 1,4-β-D-galactoside bonds in arabinogalactans. The enzyme can also be known as arabinogalactan endo-1,4-β-galactosidase, endo1,4-β-galactanase, galactanase, arabinogalactanase or arabinogalactan 4-β-D-galactanohydrolase.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one pectin acetyl esterase. Pectin acetyl esterase catalyze the deacetylation of acetyl groups in the hydroxyl groups of GaIUA residues of pectin.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one endo-pectin lyase. Endo-pectin liases (EC 4.2.2.10) catalyze the eliminative division of (1 → 4) -α-Dgalacturonan methyl ester to give oligosaccharides with 4-deoxy-6-O-methyl-α-D-galacto-4-enuronosyl groups at its non-reducing ends. The enzyme can also be known as pectin lyase, pectin trans-eliminase; endopectin lyase, polymethylgalacturonic transelliminase, pectin methyltranseliminase, pectolyase, PL, NLP or PMGL or (1 → 4) -6-O-methyl-α-D-galacturonan lyase.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one pectant lyase. Pectate liases (EC 4.2.2.2) catalyze the alternative division of (1 → 4) -α-D-galacturonan to give oligosaccharides with 4-deoxy-α-D-galacto-4-enuronosyl groups at their non-reducing ends. The enzyme can also be known as polygalacturonic transeliminase, pectic acid transeliminase, polygalacturonate lyase, endopectin methyltranseliminase, transeliminase pectate, endogalacturonate transeliminase, pectic acid lyase, pelase lyase, α-1,4-P-endo-lysa liase PPase-N, endo-α-1,4-polygalacturonic acid lyase, polygalacturonic acid lyase, pectin trans-eliminase, polygalacturonic acid trans-eliminase or (1 → 4) -α-D galacturonan lyase.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one alpha-ramnosidase. Alpha-rhamnosidates (EC 3.2.1.40) catalyze the hyrolysis of non-terminal α-L-rhamnose residues into an α-L-rhamnosidase N or α-L-rhamnosidase rhamnohydrolase.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one exo-galacturonase. Exo-galacturonases (EC 3.2.1.82) hydrolyze pectic acid from the non-reducing end, releasing

digalacturonate The enzyme can also be known as exo-poly-α-galacturonosidase, exopolygalacturonosidase or exopolygalacturanosidase.

In some additional embodiments, the heavy disclosure provides at least one GH61 and at least one galacturane 1,4-alpha-galacturonidase. Galacturane 1,4-alpha-galacturonidases (EC 3.2.1.67) catalyze a ration of the following type: (1,4, α-D-galacturonide) n + H2O = (1,4, α-D-galacturonide) or + D -galacturonate. The enzyme may also be known as ocmo poly [1-gt; 4) alpha-D-galacturonide] galacturonohydrolase, exopolygalacturonase, poly (galacturonate) hydrolase, exo-D-galacturonase, exo-D-galacturonoase, expololi-D-galacturonase or poly (1,4, α-Dgalaturonide) galacturonohydrolase.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one exopolygalacturonate lyase. Exopolygalacturonate liases (EC 4.2.2.9) catalyze the eliminative division of 4- (4deoxy-α-D-galacto-4-enuronosyl) -D-galacturonate of the pectate reducing end (ie, desterified pectin). This enzyme can also be known as disaccharide-lyso pectate, exo-lyso pectate, exopéctic acid transeliminase, exopectate lyase, exopolygalacturonic acid trans-eliminase, PATE, exo-PATE, exo-PGL or redsand disaccharide lyse end (1 → 4 ) -α-galacturonan.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one ramnogalacturonanase. Ramnogalacturonanase hydrolyzes the bond between galactorsiluronic acid and ramnopyranosyl in an endo manner in strictly alternative ramnogalactruronan structures, which consist of the disaccharide [(1,2-alpha-L-ramnoyl- (1,4) -alpha-galactosyluronic acid].

In some additional embodiments, the present disclosure provides at least one GH61 and at least one ramnogalacturonano lyase. Ramnogalacturonano liases divide α-L-Rhap- (1 → 4) -α-GalpA bonds in an endo manner into ramnogalacturonano by beta elimination.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one ramnogalacturonano acetyl esterase. Ramnogalacturonano acetyl esterases catalyze the deacetylation of the central network of alternating residues of rhamnose and galacturonic acid in ramnogalacturonan.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one ramnogalacturonan galacturonohydrolase. Ramnogalacturonano galacturonohydrolases hydrolyse galacturonic acid from the non-reducing end of strictly alternating ramnogalactacturonano structures in an exo manner. This enzyme can also be known as xylogalacturonan hydrolase.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one endo-arabinanase. Endo-arabinanases (EC 3.2.1.99) catalyze endohydrolysis of 1,5-α-arabinofuranoside bonds in 1,5-arabinanos. The enzyme can also be known as endo-arabinase, endo-1,5-α-L-arabinosidated arabinana, endo-1,5-α-L-arabinanase and endo-α-1,5-arabanase; endo-arabanase or 1,5-α-Larabinana, 1,5-α-L-arabinanohydrolase.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one enzyme that participates in the degradation of lignin in a mixture of enzymes. The depolymerization of enzymatic lignin can be performed by lignin peroxidases, manganese peroxidases, lacases and cellobiose dehydrogenases (CDH), often working in synergy. These extracellular enzymes are often referred to as "lignin modifying enzymes" or "EMLs." Three of these enzymes comprise two peroxidases that contain glycosylated hemoglobin: lignin peroxidases (LIP); Mn-dependent peroxidase (MNP); and copper containing peroxidase (LCC):

In some additional embodiments, the present disclosure provides at least one GH61 and at least one laccase. Lacases are oxidase enzymes that contain copper found in many plants, fungi and microorganisms. Lacases are enzymatically active in phenols and similar molecules and perform an oxidation of electrons. Lacases can be polymeric and the enzymatically active form can be a dimer or trimer.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one Mn-dependent peroxidase. The enzymatic activity of Mn-dependent peroxidase (MnP) is dependent on Mn2 +. Without being bound by any theory, it has been suggested that the main role of this enzyme is to oxidize Mn2 + to Mn3 + (See, for example, Glenn et al., Arch. Biochem. Biophys., 251: 688-696 [1986]). Subsequently, the phenolic substrates are oxidized by the generated Mn3 +.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one lignin peroxidase. Lignin peroxidase is an extracellular hemoglobin that catalyzes the oxidative depolymerization of dilute solutions of polymeric lignin in vitro. Some of the LiP substrates, most notably 3,4-dimethoxybenzyl alcohol (veratril alcohol, VA), are redox active compounds that have been shown to act as redox mediators. VA is a secondary metabolite produced at the same time as LiP through cultures

ligninolytics of P. chrysosporium and without being bound to any theory, have been proposed to function as a physiological redox mediator in LiP-catalyzed oxidation of lignin in vivo (See, for example, Harvey, et al., FEBS Lett., 195-242-246 [1986])

In some additional embodiments, the present disclosure provides at least one GH61 and at least one protease, amylase, glucoamylase and / or a lipase that participates in cellulose degradation.

As used herein, the term "protease" includes enzymes that hydrolyze peptide bonds (peptidases), as well as enzymes that hydrolyze bonds between peptides and other fractions, such as sugars (glycopeptidases). Many proteases are characterized under EC 3.4, and are suitable for use in the invention. Some specific types of proteases include cysteine proteases that include pepsin, papain and serine proteases that include chymotrypsins, carboxypeptidases and metalloendopeptidases.

As used herein, the term "lipase" includes enzymes that hydrolyse lipids, fatty acids and acylglycerides, including phosphoglycerides, lipoproteins, diacylglycerols and the like. In plants, lipids are used as a structural component to limit water loss and infection of pathogens. These lipids include waxes derived from fatty acids, as well as cutin and suberine.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one expansin or an expansin-like protein, such as swollenin (See, for example, Salheimo et al., Eur. J. Biochem., 269: 42002- 4211 [2002]) or a swollenin-like protein. Expansins are involved in the release of the cell wall structure during plant cell growth. Expansins have been proposed to disrupt hydrogen bonding between cellulose and other cell wall polysaccharides without having hydrolytic activity. In this way, it is thought that they allow the sliding of cellulose fibers and the increase of the cell wall. Swollenin, an expansin-like protein contains a Family 1 domain of the N-Terinal Carbohydrate Link Module (CBD) and a C-terminal expansin type domain. In some embodiments, an expansin type protein or a swollenin type protein comprises one or both of said domains and / or disrupts the cell wall structure (such as disrupting the cellulose structure), optionally without producing detectable amounts of reducing sugars.

In some additional embodiments, the present disclosure provides at least one GH61 and at least one polypeptide product of a cellulose, scaffold or scaffold-like protein, for example CipA or CipC of Clostridium thermocellum or Clostridium cellulocitycum respectively. Scaffolds and cellulose integrating proteins are multi-functional sub-units that can organize cellulolytic sub-units in a multi-enzyme complex. This is achieved through the interaction of two complementary domain classes (that is, a scaffold cohesion domain and a molecular coupling domain in each enzyme unit). The scaffolding sub-unit also has a module that bonds with cellulose that mediates the union of cellulose with its substrate. A scaffold or protein that integrates cellulose for the purposes of this invention may comprise one or both of these domains.

In some embodiments, the present disclosure provides a GH61 and at least one cellulose-induced protein or modulating protein, for example as encoded by the cip1 cip2 gene or similar Trichoderma reesei genes (See, for example, Foreman et al., J Biol. Chem. 278: 31988-31997 [2003]).

In some additional embodiments, the present disclosure provides at least one GH61 and at least one member of each of the polypeptide classes described above, several members of a polypeptide class, or a combination of these polypeptide classes to provide mixtures of Enzymes suitable for various uses.

In some disclosures of the invention, the enzyme mixture comprises other types of cellulases, selected but not limited to cellobiohydrolase, endoglucanase, β-glucosidase and glycoside hydrolase protein 61 (GH61). These enzymes can be wild type or recombinant enzymes. In some embodiments, celloiodiodollase is a type 1 cellobiohydrolase (eg, T. reesei cellobiohydrolase I). In some embodiments, the endoglucanase comprises a catalytic domain derived from the catalytic domain of a Streptomyces avermitilis endoglucanase (See, for example, United States Patent Application Publication No. 2010/0267089). In some embodiments, the at least one cellulase is derived from Acidothermus cellulolyticus, Thermobifida fusca, Humicola grisea, Myceliophthora thermophila, Chaetomium thermophilum, Acremonium sp., Thielavia sp, Trichoderma reesei, Aspergillus sp., Or a Chrysosporium sp. The cellulase enzymes of the cellulase mixture work together resulting in a de-crystallization and hydrolysis of the cellulose of a biomass substrate to produce fermentable sugars, such as, but not limited to, glucose.

Some cellulase mixtures for efficient enzymatic hydrolysis are known (See, for example, Viikari et al., Adv. Biochem. Eng. Biotechnol., 108: 121-45 [2007]; and US Patent Application Publications US Numbers 2009/0061484, US 2008/0057541 and US 2009/0209009). In some embodiments, mixtures of purified enzymes that occur naturally or recombinantly are combined with cellulosic raw material or

a cellulose hydrolysis product. Alternatively or in addition to one or more cell populations, each producing one or more naturally occurring or recombinant cellulases, they are combined with cellulosic raw material or a cellulose hydrolysis product.

In some embodiments, the enzyme mixture comprises commercially available purified cellulases. Commercial cellulases are known and available (for example, C2730 cellulase from Trichoderma reesei ATCC No. 25921 available from Sigma-Aldrich, Inc .; and C9870 ACCELLERASE® 1500, available from Genencor).

XIII Cellulosic substrate

Cellulosic substrates may be derived from any material containing cellulose, such as biomass derived from plants, animals or microorganisms, and may include agricultural, industrial or forestry wastes, industrial or municipal wastes, and land and aquatic crops that grow for energy purposes. "Cellulosic substrates" broadly encompass any living or dead material containing a polysaccharide substrate, including but not limited to, cellulose, starch, other forms of long chain carbohydrate polymers, and mixtures of such sources. It may or may not be assembled entirely or mainly from glucose or xylose, and may also optionally contain several other pentose or hexose monomers. Xylose is an aldopentose that contains five carbon atoms and an aldehyde group. It is the precursor of hemicellulose, and is often a main constituent of biomass.

Cellulose substrates are often provided as lignocellulose feedstocks that can be processed prior to cellulase hydrolysis. As used herein, the terms "lignocellulose feedstock" refer to any type of plant biomass as, but not limited to, cultivated crops (eg, herbs, including C4 herbs such as rod grass, spartin, rye, miscanthus, ribbon weed or any combination thereof), residues that process sugar, for example, but not limited to, bagasse (for example, sugarcane bagasse, beet pulp or a combination of them), agricultural residues (for example, stubble of soya, corn stubble, corn fiber, rice straw, sugar cane straw, rice, rice pods, barley straw, corn cobs, wheat straw, canola straw, oat straw, oat pods , corn fiber, hemp, flaxseed, sisal, cotton or any combination thereof), fruit pulp, vegetable pulp, distiller grains, forest biomass (e.g. wood, wood pulp, paper pulp, fiber pulp recycled wood, sawdust, hardwood, com or poplar wood, softwood, or a combination of them). In addition, in some embodiments, the lignocellulose feedstock comprises cellulosic waste material and / or forest waste materials, including but not limited to, waste from paper and pulp processing, newspaper, cardboard and the like. Biomass can also comprise transgenic plants that express ligninase and / or cellulase enzymes (US 2008/0104724 A1).

In some embodiments, the lignocellulosic raw material comprises a kind of fiber, while in some alternative embodiments, the cellulosic raw material comprises a mixture of fibers that originate from different lignocellulosic raw materials. In some other embodiments, the lignocellulosic raw material comprises fresh lignocellulosic raw material, partially dry lignocellulosic raw material, completely dry lignocellulosic raw material, and / or combinations thereof. In some embodiments, the lignocellulosic raw material comprises cellulose in an amount greater than about 20%, more preferably greater than 30%, more preferably greater than 40% (w / w). For example, in some embodiments, the lignocellulosic material comprises from about 20% to about 90% (w / w) cellulose, or any amount between them, although in some embodiments, the lignocellulosic material comprises less than about 19%, less than approximately 18%, less than approximately 17%, less than approximately 16%, less than approximately 15%, less than approximately 14%, less than approximately 13%, less than approximately 12%, less than approximately 11%, less than approximately 10 %, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5% cellulose (w / w). In addition, in some embodiments, the lignocellulosic feedstock comprises lignin in an amount greater than about 10%, more typically in an amount greater than about 15% (w / w). In some embodiments, the lignocellulosic raw material comprises small amounts of sucrose, fructose and / or starch.

Lignocellulosic raw material generally undergoes a reduction in size by methods that include, but are not limited to, grinding, spraying, agitation, crushing, compression / expansion or other types of mechanical action. The reduction of size by mechanical action can be carried out by any type of equipment adapted for this purpose, for example, but not limited to, hammer grinders, choppers with buckets, presses with rolls, refiners and blenders. In some embodiments, at least 90% by weight of the particles produced from the size reduction have lengths less than between about 1/16 and about 4 (the measurement may be by a volume or an average weight length). In some embodiments, the equipment used to reduce the particle size is a hammer grinder or a crusher. After the reduction in size, the raw material is liquefied in water, as this facilitates the pumping of the raw material. In some embodiments, lignocellulosic raw material of particle size less than about 6 inches does not require size reduction. Biomass can optionally be pretreated to increase the

susceptibility of cellulose to hydrolysis through chemical, physical and biological pre-treatments (such as steam explosion, pulp reduction, spraying, acid hydrolysis, exposure to solvents and the like, as well as combinations thereof).

In some embodiments, the raw material is liquefied before pretreatment. In some embodiments, the consistency of the raw material sludge is between about 2% and about 3% and more typically between about 4% and about 5%. In some embodiments, the sludge is subjected to a soaking operation in water and / or acid before pretreatment. In some embodiments, the sludge is drained using any suitable method to reduce the use of steam or chemicals before pretreatment. Examples of drainage devices include, but are not limited to, pressurized presses with screws (See, for example WO 2010/022511), pressurized filters or extruders.

As used herein, the terms "pretreated lignocellulosic raw material" and "pretreated lignocellulose" refer to lignocellulosic raw materials that have undergone physical and / or chemical processes to make the fiber more accessible and / or receptive to the actions of cellulolytic enzymes. Thus, "pretreated lignocellulosic raw material" is an example of a "pretreated cellulosic substrate." In some embodiments, the pretreatment is performed to hydrolyze hemicellulose and / or a part thereof present in the lignocellulosic matter to monomeric pentose and hexose sugars (for example, xylose, arabinose, mannose, galactose and / or any combination of the same). In some embodiments, the pre-treatment is performed so that almost complete hydrolysis of hemicellulose and a small amount of cellulose or glucose conversion occurs. In some embodiments, an acid concentration in the aqueous sludge of from about 0.2% (w / w) to about 2% (w / w) is typically used.

or any intermediate amount, for the treatment of lignocellulosic raw material. Any suitable acid finds use in these methods, including but not limited to, hydrochloric acid, nitric acid and / or sulfuric acid. In some embodiments, the acid used during the pre-treatment is sulfuric acid. Vapor explosion is a method of pre-treating with raw material acid (See, for example, US Patent No. 4,461,648). Another method of pretreating the raw material sludge includes continuous pretreatment (that is, the lignocellulosic raw material is pumped through a reactor continuously). These methods are well known to those skilled in the art (See, for example, U.S. Patent No. 7,754,457.

In some embodiments, alkali is used in pre-treatment. Unlike pre-treatment with acid, pre-treatment with alkali may not hydrolyze the hemicellulose component of the raw material. Rather the alkali reacts with acidic groups present in hemicellulose to open the surface of the substrate. In some embodiments, the addition of alkali alters the cellulose crystal structure to make it more susceptible to hydrolysis. Examples of alkali that find use in pre-treatment include, but are not limited to, ammonia, ammonium hydroxide, potassium hydroxide and sodium hydroxide. One method of pre-treatment with alkali is ammonia suspension explosion, ammonia fiber explosion or ammonia fiber expansion (“AFEX” Process; See, for example, United States Patents Numbers 5,171,592; 5,037,663; 4,600,590; 6,106,888; 4,356,196; 5,939,544; 6,176,176; 5,037,663 and 5,171,592). During this process, the lignocellulosic raw material contacts ammonia or ammonium hydroxide in a pressure vessel for a sufficient time that allows the ammonia or ammonium hydroxide to alter the crystal structure of the cellulose fibers. Then the pressure is quickly reduced, allowing the ammonia to flash or heat and explode the cellulose fiber structure. In some embodiments, the flashed ammonia is then recovered using methods known in the art. In an alternative method, a pre-treatment with diluted ammonia is used. The method with the pre-treatment of diluted ammonia uses more dilute solutions of ammonia or ammonium hydroxide than AFEX (See, for example, WO 2009/045651 and US 2007/0031953). This pre-treatment process may or may not produce monosaccharides.

An additional pre-treatment process for use in the present invention includes chemical treatment of the raw material with organic solvents, in methods such as those using organic liquids in pre-treatment systems (See, for example, US Pat. No. 4,556,430). These methods have the advantage that low boiling liquids can be easily recovered and reused. Other pretreatments, such as the Organosolv ™ process, also use organic liquids (See, for example, U.S. Patent No. 7,465,791.). Subjecting the raw material to pressurized water may also be a suitable method of pre-treatment (See, for example, Weil et al., (1997) Appl. Biochem. Biotechnol. 68 (1-2): 21-40).

In some embodiments, the pretreated lignocellulosic feedstock is processed after pretreatment by any of several stages, such as dilution with water, washing with water, damping, filtration.

or centrifugation or any combination of these processes, before enzymatic hydrolysis, as will be familiar to those skilled in the art. The pre-treatment produces a pre-treated raw material composition (for example, a "pre-treated raw material sludge") containing a soluble component that includes sugars resulting from the hydrolysis of hemicellulose, optionally acetic acid and other inhibitors , and solids that include raw material and non-hydrolyzed lignin. In some embodiments, the soluble components of the pre-treated raw material composition are separated from the solids to produce a soluble fraction. In some embodiments, the soluble fraction, including sugars released during pre-treatment and other soluble components (eg, inhibitors), is then sent to fermentation. However, in some embodiments where hemicellulose is not hydrolyzed effectively during pre-treatment, one or more stages are included.

additional (for example, an additional stage of hydrolysis and / or stages of enzymatic treatment and / or additional treatment with alkali and / or acid) to produce fermentable sugars. In some embodiments, the separation is performed by washing the pre-treated raw material composition with an aqueous solution to produce a wash jet and a solid stream comprising the non-hydrolyzed pretreated raw material.

Alternatively, the soluble component is separated from the solids by subjecting the pre-treated raw material composition to a solid-liquid separation, using any suitable method (e.g., centrifugation, microfiltration, plate filtration, cross flow filtration, filtration of pressure, vacuum filtration, etc.). Optionally, in some embodiments, a solid-liquid separation step is incorporated. In some embodiments, separated solids containing cellulose then undergo enzymatic hydrolysis with cellulase enzymes in order to convert cellulose into glucose. In some embodiments, the pretreated raw material composition is introduced into the fermentation process without separation of the solids contained. In some embodiments, non-hydrolyzed solids are subjected to enzymatic hydrolysis with cellulase enzymes to convert cellulose into glucose after the fermentation process. The pretreated lignocellulose is subjected to enzymatic hydrolysis with cellulase enzymes.

The pre-treatment produces a pre-treated raw material composition (eg, pre-treated raw material sludge) that contains a soluble component that includes the sugars resulting from the hydrolysis of hemicellulose, optionally acetic acid and other inhibitors, and solids which include raw material and non-hydrolyzed lignin.

Soluble components of the pre-treated raw material composition can be separated from solids to produce a soluble fraction for use in the saccharification reaction.

The separation can be done by washing the pre-treated raw material composition with an aqueous solution to produce a wash jet, and a solid stream comprising non-hydrolyzed pre-treated raw material. Alternatively, the soluble component is separated from solids by subjecting the pre-treated raw material composition to a solid-liquid separation, using methods such as centrifugation, microfiltration, plate filtration, cross flow filtration, pressure filtration and / or filtration. of emptiness. Optionally, a washing step in the solid-liquid separation can be incorporated. Separated solids containing cellulose can then undergo enzymatic hydrolysis with cellulase enzymes for conversion of cellulose to glucose.

Properly prepared lignocellulose can be subjected to enzymatic hydrolysis using one or more cellulase enzymes in the presence of one or more GH61 proteins or preparations according to this invention.

Hydrolysis of hemicellulose and cellulose components of a lignocellulosic raw material produces a lignocellulosic hydrolyzate comprising xylose and glucose. Other sugars typically present include galactose, mannose, arabinose, fucose, rhamnose or a combination thereof. Regardless of the means to hydrolyze lignocellulosic raw material (complete acid hydrolysis or chemical pre-treatment with or without subsequent enzymatic hydrolysis), xylose and glucose generally form a large proportion of the sugars present.

If the lignocellulosic hydrolyzate is a hemicellulose hydrolyzate resulting from the acid pre-treatment, xylose will be the predominant sugar and smaller amounts of glucose will be present, because a modest amount of cellulose hydrolysis typically occurs during the pre-treatment. In this case, the xylose can form between about 50 and about 90% by weight of the total carbohydrate content of the cellulose hydrolyzate. If lignocellulosic hydrolyzate results from sequential pre-treatment and enzymatic hydrolysis of lignocellulosic feedstock (that is, without a solids separation stage after pre-treatment), the xylose can form between about 30 and about 50% by weight of the total carbohydrate content. The relative amount of xylose present in the lignocellulosic hydrolyzate will depend on the raw material and the pretreatment used.

Soluble components of the hydrolyzed substrate can be separated from solids to produce a soluble fraction. The soluble fraction (including sugars released during hydrolysis, and sometimes inhibitors) can then be used for fermentation. If hemicellulose is not hydrolyzed effectively during pretreatment, it may be desirable to include an additional stage of hydrolysis or stages with enzymes or an additional treatment with alkali or acid to produce fermentable sugars.

XIV: Fermentation of sugars

Fermentable sugars produced in saccharification reactions using GH61 proteins of the disclosure can be used to produce various end products of interest.

In some embodiments, sugars are used in a fermentation process to produce final products. The term "fermentation" is widely used to refer to the cultivation of a microorganism or a culture of microorganisms that use simple sugars, such as fermentable sugars, as a source of energy for

Get a desired product. In a different embodiment, cellulosic biomass can be treated with a composition of this invention to prepare animal feed.

The end products include alcohols (for example, ethanol, butanol), acetone, amino acids (for example, glycine and lysine), organic acids (for example, lactic acid, acetic acid, formic acid, citric acid, oxalic acid, 3- acid hydroxypropyinoic acid, acrylic acid, succinic acid, malic acid, fumaric acid or uric acid), glycerol, diols (such as 1,3 propanediol or butanediol), hydrocarbons with 1-20 carbon atoms (for example, long chain esters), alcohols of sugar (for example, xylitol), fatty alcohols and β-lactam and other end products.

Any microorganism can be used to convert sugar in the sugar hydrolyzate to ethanol or other fermentation products. These include yeasts of the genus Saccharomyces, Hansenula, Pichia, Kluyveromyces and Candida. Commercially available yeasts, such as Turbo yeast, Ethanol Red® Safdistil®, Thermosacc®, Fermiol®, Fermivin® or Superstart ™, can be used.

Yeast can be genetically produced for hexose and pentose sugars to a final product, including but not limited to ethanol. Alternatively, the yeast can be a strain that has been able to ferment xylose and glucose by one or more non-recombinant methods, such as adaptive evolution or mutagenesis and random selection. For example, fermentation can be performed with recombinant Saccharomyces yeast. Recombinant yeast is a strain that has been able to ferment xylose by incorporating recombinant genes that encode xylose reductase (XR) and xylitol dehydrogenase (XDH) (US Patents 5,789,210, 5,866,382, 6,582,944 and 7,527. 927 and EP 450 530), and / or genes encoding one or more xylose isomerase (XI) (US Patents 6,475,768 and 7,622,284). In addition, the modified yeast strain can overexpress an endogenous or heterologous gene encoding xylukinase (XK): another yeast can ferment hexose and pentose sugars to at least one final product, including but not limited to, ethanol, as a yeast of the Hansenula genera , Pichia, Kluyveromyces and Candida (WO 2008/130603).

A typical temperature range for the fermentation of a sugar to ethanol using Saccharomyces spp. it is between approximately 25 ° C and approximately 37 ° C, although the temperature may be higher (up to 55 ° C) if the yeast has been modified naturally or genetically to be thermostable. The pH of a typical fermentation using Saccharomyces spp. it is between about 3 and about 6, depending on the optimum pH of the fermentation microorganism. Hydrolyzed sugar can also be supplemented with additional nutrients necessary for the growth and fermentation of the microorganism for fermentation. For example, yeast extract, specific amino acids, phosphate, nitrogen sources, salts, trace elements and vitamins (Verduyn et al., 1992, Yeast 8 (7): 501-170, Jørgensen, 2009, Appl Biochem Biotechnol, 153 : 44-57 and Zhao et al., 2009, Journal of Biotechnology, 139: 55-60). Typically, fermentation is performed under anaerobic conditions, although aerobic or microaerobic conditions can also be used.

Thus, the invention provides processes for producing a fermentation product, where the method comprises: providing the recombinant host cells as provided herein, a fermentation medium comprising fermentable sugars such as glucose and / or xylose; and contacting the fermentation medium with the recombinant fungal host cells under suitable conditions to generate the fermentation product. In some embodiments, the processes further comprise the step of recovering the fermentation product. In some additional embodiments, the fermentation stage is performed under micro-aerobic or aerobic conditions. In some embodiments, the fermentation stage is performed under anaerobic conditions. In some embodiments, the fermentation medium comprises product of a saccharification process.

The GH61 proteins and cellulases of the present disclosure can be used in any method used to generate alcohols or other biofuels from cellulose, and are not necessarily limited to those described herein. Two commonly used methods are the separate saccharification and fermentation (SHF) method (see, Wilke et al., Biotechnol. Bioengin. 6: 155-75 (1976)) or the simultaneous saccharification and fermentation (SSF) method disclosed by example in U.S. Patent Nos. 3,990,944 and 3,990,945.

The SHF method of saccharification of the present invention comprises the steps of contacting a GH61 protein cellulase with a cellulase containing substrate to enzymatically break down the cellulase into fermentable sugars (e.g., monosaccharides such as glucose), contacting the fermentation sugars with a microorganism alcohol producer to produce alcohol (eg, ethanol or butanol) and recover the alcohol. In some embodiments, the consolidated bioprocessing method (CBP) can be used, where the production of host cellulase is simultaneous with saccharification and fermentation, either of a host or of a mixed culture.

In addition to SHF methods, an SSF method can be used. In some cases, the SSF methods result in greater alcohol production efficiency than that produced by the SHF method (Drissen et al., Biocatalysis and Biotranformation 27: 27-35 (2009)). A disadvantage of SSF over SHF is that higher temperatures are needed for SSF than for SHF.

In one embodiment, the present invention uses cellulase polypeptides that have greater thermostability.

than a wild type cellulase.

EXAMPLES

5

Example 1. Identification of GH61 proteins in M. tehrmophila

The genomic sequence of a wild-type fungal strain of M. thermophila was obtained. The

10

entire genome to identify and evaluate protein coding regions. Twenty-four endogenous proteins were selected for the strain M. thermophila based on factors that include the presence of sequence motifs.

glycohydrolase family 61 (GH61) (Pfam domains). Pfam domains were identified using the algorithm

“PFAM V.24” software, developed by Welcome Trust Sanger Institute (Henrissat et al., 1995, Proc Natl Acad Sci

USA 92: 7090-94).

fifteen

TABLE 1 provides the GH61 pre-protein sequence (SEQ ID NO: 1) and the predicted secreted form

(mature) (SEQ ID NO: 2). The mature protein was designated "GH61a". TABLE 2 provides the sequence of other

GH61 pre-proteins, with the predicted native signal peptide underlined. Two of the proteins in TABLE 2 do not

They predict by having signal peptides.

twenty

In TABLE 3, below, the sequential numbering and domain structure analysis of

the corresponding polynucleotides.

25

30

35

40

Four. Five

fifty

55

TABLE 3: Sequence numbering and domain analysis

Protein Designation SEQ ID Nucleic Acid PFAM Protein Laboratory Domain NO SEQ ID NO

GH61a 1 31 GH61 — CBM_1 GH61l 3 32 Chitin_bind_3 - GH61

10 GH61m 4 33 GH61 GH61n 5 34 GH61 GH61o 6 35 GH61 — GH61-CBM_1 GH61p 7 36 GH61-GH61

15 GH61q 8 37 GH61

GH61r 9 38 GH61

GH61s 10 39 GH61 20

GH61t 11 40 GH61 GH61u 12 41 GH61 GH61v 13 42 GH61

25 GH61w 14 43 GH61 GH61x 15 44 GH61 GH61b 16 45 GH61 GH61c 17 46 GH61

30 GH61d 18 47 GH61

GH61e 19 48 GH61

GH61f 20 49 GH61 — CBM_1 35

GH61g 21 50 GH61 — CBM_1 GH61h 22 51 GH61 GH61i 23 52 GH61

40 GH61j 24 53 GH61 GH61k 25 54 GH61 GH61p2 26 55 GH61 - GH61 GH61q2 27 56 GH61

45 GH61r2 28 57 GH61

GH61t2 29 58 GH61

GH61e2 30 59 GH61 50

SEQ ID NO: 7 has a second domain GH61 (GH61-GH61), SEQ ID Nos: 1, 20, 21 have the structure GH61CBM1 (where "CBM1" is a module 1 that binds carbohydrate), SEQ ID NO: 6 It has the structure GH61

55 GH61-CBM1, SEQ ID Nos: 4, 5, 8-19 and 22-25 have the structure GH61, SEQ ID NO: 3 has the structure Chitin_bind_3-GH61 (where "Chitin_bind_3" is module 3 that links with chitin) . SEQ ID Nos: 26-30 are alternative sequences corresponding to the genes encoding SEQ ID Nos: 7-9, 11 and 19, respectively.

Example 2. Recombinant expression of GH61 proteins

Among the GH61 proteins identified in Example 1, certain examples were selected for expression based on the predicted structure and functional aspects, as if the protein would be secreted from the cell, and its domain structure.

The six GH61 proteins listed in the following table were individually cloned into an expression vector under the control of a CHI promoter (constitutive of the target cell) and transformed into a strain of M. thermophila designated "CF-405" that has been adapted to be deficient in endogenous cellulase production.

TABLE 4: Recombinantly expressed GH61 proteins

Laboratory designation

SEQ ID NO protein

GH61a

2

GH61o

6

GH61v

13

GH61x

fifteen

GH61b 16 GH61e 19, 30

Transformed cells expressing recombinant GH61 protein were selected and seeded cultures were prepared. The progeny cells were cultured for 5 days, and the broth containing the secreted GH61 protein ("GH61 broth") was collected.

Example 3. GH61 protein cellulase enhancer activity

The broth was collected from cells expressing the GH61 protein comprising SEQ ID NO: 2 (Example 2), and the level of recombinant GH61 was quantified by SDS-PAGE. Cellulase assays were performed to determine if the addition of GH61 protein improved the hydrolysis of cellulosic material by cellulase enzymes.

The culture broth was collected from a culture of a strain of M. thermophila designated "CF-402" that overexpends β-glucosidase. Cellulase digestion assays were performed using the broth with and without added GH61. The reactions were done in 96 Costar deep well plates in a total reaction volume of approximately 80 microliters. The reactions were performed at 55 ° C using substrate preparations containing wheat straw cellulose that had been pretreated under acidic conditions (hereafter referred to as "pretreated wheat straw"). 8.1 mg of broth protein per gram of substrate (0.82%) was added to each sample. In addition, the samples had 0 (control), 0.22%, 0.44% or 0.66% GH61 broth protein added (final total concentration of 0.81%, 1.03%, 1.25 broth protein % or 1.47%). In control wells without added GH61, water was used to adjust the volume so that the substrate load was the same in all wells. In other experiments, 6-10 mg of broth protein per gram of substrate (0.6-1%) was added to each sample, at a final total broth protein concentration of 0.6-1.7%.

FIGURE 1 shows additional glucose production over the control (broth without added GH61) after 48 hours of incubation. In FIGURE 1 (A), the percentage of improved production over the control is shown. In FIGURE 1 (B), the data is plotted to show total glucose production.

Example 4. Overexpression of GH61a in a CXP strain

Construction of recombinant GH61a genes

Genomic DNA from strain M. thermophila designated "CF-409" was isolated. This cpa endogenously produces endoglucanase, β-glucosidases, Type 1 cellobiohydrolase, and Type 2 cellobiohydrolase. The procedure was as follows. The inoculum of the daughters was shaded in a culture medium and allowed to grow for 72 hours at 35 ° C. The micellar mat was collected by centrifugation, washed and 50 µL of DNA extraction buffer (200 M Tris pH 8.0; 250 mM NaCl; 125 mM EDTA; .5% SDS) was added. The mycelia were ground with a conical grinder, re-extracted with 250 µL of extraction buffer and the suspension was centrifuged. The supernatant was transferred to a new tube containing 300 µL isopropanol. The DNA was collected by centrifugation, washed twice with 70% ethanol and re-dissolved in 100 µL of deionized water.

The GH61a DNA sequence was amplified from CF-409 cells using primers PchiC1GH61a_F and TcbhC1GH61a_R. PCR reaction was performed using Phusion Polymerase, at 98 ºC for 30 ’, followed by 35 cycles of 98 ºC for 10’, 72 ºC for 1 ’and the final extension at 72 ºC for 5’. The resulting product was cloned in the 3 'pC1DX20PhR vector to the chi1 promoter to create an expression vector that expressed transcription of GH61 protein under the control of the chi1 promoter (pC1DX20PhR-GH61a) using In-fusion cloning technique (In cloning kit -Fusion Advantage ™ PCR with cloning enhancer, Clontech Cat. No. 639617 according to the manufacturer's instructions).

PchiC1GH61a_F tacttcttctccaccATGTCCAAGGCCTCTGCTCT SEQ ID NO: 69 TcbhC1GH61a_R ggatccgaattcttaTTACAAACACTGGGAGTACCA SEQ ID NO: 70

Preparation of protoplast for CXP transformation

M. thermophila cells ("CF-405", an Alp1 removed strain) were inoculated into 100 mL of growth medium in a 500 mL Erlenmeyer flask using 106 spores / ml. The culture was incubated for 24 hours at 35 ° C, 250 rpm. To harvest the mycelium, the culture was filtered on a sterile Myracloth ™ filter (Calbiochem) and washed with 100 mL solution 1700 mosmol NaCl / CaCl2 (0.6 M NaCl, 0.27 M CaCl2 · H2O). The washed mycelium was transferred to a 50 mL tube and weighed. Cailase (20 mg / gram mycelium) was dissolved in 1700 mosmol NaCl / CaCl2 and sterilized with UV for 90 seconds. 3 ml of Caylase sterile solution was added to the washed mycelium containing the tube and mixed. An additional 15 mL of 1700 mosmol NaCl / CaCl2 solution was added to the tube and mixed.

The mycelium / Caylase suspension was incubated at 30 ° C, 70 rpm for 2 hours. The protoplasts were harvested by filtering them through a sterile Myracloth filter in a sterile 50 mL tube. 25 mL of cold STC was added to the flow and rotated at 2720 rpm for 10 min at 4 ° C. The granule was resuspended in 50 mL sTC (1.2 M sorbitol, 50 mM CaCl2 · H2O, 35 mM NaCl, 10 mM Tris-HCl) and centrifuged again. After the washing steps, the granule was resuspended in 1 mL STC.

Transformation

At the bottom of a sterile 5 mL tube, plasmid DNA was placed in a pipette and 1 µL of autrin-carboxylic acid and 100 µL of protoplast were added. The content was mixed and the protoplast with the DNA was incubated at room temperature for 24 min. 1.7 ml of PEG4000 solution (60% PEG4000 [polyethylene glycol, 4000 Daltons molecular average weight], 50 mM CaCl2 · H2O, 35 mL NaCl, 10 mM Tris-HCL) were added and mixed thoroughly. The solution was kept at room temperature for 20 min. The tube was filled with STC, mixed and centrifuged at 2500 rpm for 10 min at 4 ° C. STC was poured and the granule was resuspended in the rest of STC and plated on plates of minimum medium. The plates were incubated for 5 days at 35 ° C. The colonies were stained again and checked for the presence of an integrated plasmid. Several isolates were selected and tested for GH61a expression.

The transformation was performed in CF405 strains. GH61a transformants were tested for overexpression of GH61 in SDS-PAGE for the presence of the extra band that was confirmed by MS-MS analysis.

Example 5. Purification of GH61 proteins from cellulase supernatant

In this experiment, the GH61 protein activity was fractionated from the culture supernatant of a strain of M. thermophila designated "CF-401". CF-401 was derived from CDXF which has a CDH1 and CDH2 gene deletion. This strain endogenously produces endoglucanase, β-glucosidases, Type 1 cellobiohydrolase, and Type 2 cellobiohydrolase.

To prepare for chromatography, the complete CF-401 cellulase supernatant was clarified by centrifugation at 12,000 rpm for 35 minutes. The supernatant was then filtered through a 0.22 µm PES ™ membrane, which was then concentrated and exchanged with buffer in a 25 mM bis-tris buffer, pH 5.7. Saturated ammonium sulfate in 25 mM bis-tris was added to a final concentration of ≈50 g / L protein in 0.9 M ammonium sulfate and 20-25 mM bis-tris. This solution was immediately loaded onto a FF Phenyl (High Sub) column (a fast-flow column packed with Phenyl-Sepharose ™) and rinsed with 0.9 M ammonium sulfate in 25 mM bis-tris until the A280 fell near the starting point. The protein was eluted with a gradient of 0.9 M ammonium sulfate in 25 mM bis-tris to 0 M ammonium sulfate in 25 mM bis-tris, on approximately 10 column volumes. The fractions were collected (25 in this case) according to the chromatogram peaks (A280). To inhibit simultaneous growth, NaN3 was added to all fractions to a final concentration of 0.05%. After maintaining an SDS-PAGE gel in each fraction, similar fractions were combined to create groups with a minimum set of components. Here, of the 25 fractions produced, 11 groups resulted. To prepare the next column, each group was concentrated to ≈150 mL using a tangential flow filtration unit equipped with 50 kDa MWCO PES ™ membranes. The volume of the protein solution was then returned to 500 mL with DI water, and concentrated again to ≈150 mL; This step was repeated 5x and the solution was subjected to buffer exchange effectively.

Each of the 11 groups derived from CF-401 was further fractionated with a Q column (quaternary ammonium ion exchange resin). After quantification of total protein using a BCA (bicinconinic acid) assay, solutions of 50 g / L protein in 10 mM bis-tris pH 7.5 buffer for each group were prepared. After application to the column, the resin was washed with 10 mM bis-tris, pH 7.5 until A280 fell near the starting point. The bound protein was then eluted with 1 M NaCl in 10 mM Bis-Tris, pH 7.5 using a gradual gradient (5% for 10 minutes), and maintained at that concentration until the protein peak

It started to go down continuously. For analysis, an SDS-PAGE gel was maintained in each fraction, and fractions with similar compositions were pooled. Each of these second phase groups was then desalted / concentrated with tangential flow filtration.

In total, 79 groups of enzymes derived from CF-401 were prepared in this assay. To aid in loading in the saccharification assay, each total protein for each group was measured using the BCA protein assay.

Example 6. Use of GH61 proteins to promote saccharification

The fractionated GH61 enzymes according to the previous example were tested in saccharification in the presence of complete cellulase systems obtained from a cell strain designated "CF-404". These cells were derived from strain CF-401 and overexpressed β-glucosidase. As a control experiment, 0.21% (generally 0.1-4%) of CF-404 was added to 0.61% (generally 0.6-1.5%) of CF-401. Thus, the total protein load was the same in all reactions. These mixtures were then incubated with 110 g / L glucan (pretreated wheat straw) at 55 ° C, pH 4.6-5 for 53 hours. At the end of the experiment, the reactions were cooled by the addition of 10 mM sulfuric acid. For glucose analysis, the samples were analyzed using an Agilent HPLC 1200 equipped with HPX-87H ion exclusion column (300 mM x 7.8 mM) with 5 mM H2SO4 as a mobile phase at a flow rate of 0.6 ml / min at 65 ° C. The glucose retention time was 9.1 minutes.

An example demonstrating improved glucose production is shown in Table 5.

TABLE 5: Enhanced saccharification using fractions of GH61

0.21% GH61

Glucose produced SEQ ID NO

complementing 0.61%

(g / L) correspondent

CF-404

GH61

> 30 twenty

GH61

> 35 2

GH61

> 35 13

GH61

> 30 7

GH61

> 30 21, 26

GH61

> 35 2. 3

Control (CF-404)

28.9 ± 0.66

The partial protein sequence was obtained from mass spectrometry and compared to the sequences encoding proteins identified in the genome sequence of M. thermophila (Example 1) using the BIFX alignment software available from Bioinformatics Organization, Hudson MA. Concordance with the known sequences of M. thermophila is shown in the table above.

Example 7. Minimum combination of protein to convert cellulose into glucose

The enzymes of M. thermophila, CBH1 CBH2 were combined with various combinations of the GH61 proteins; GH61a, GH61f and GH61p, and were tested at various relative concentrations for the ability to convert glucan to glucose. The supernatant of strain CF-401 (comprising cellulases and GH61 proteins) were also tested for comparison. For 110 g / L glucan, the maximum possible glucose productions are approximately 135 to 145 g / L:

The saccharification reactions were carried out in 110 g / L glucan load of pre-treated wheat straw at pH 5.0 at a temperature of 55 ° C at 950 rpm in a total volume of 95 µL in 96 deep high well plates performance (HTP). 81 g / L xylose and 128 mM acetate were added to the pre-treated wheat straw. Excessive β-glucosidase (in relation to glucan) was also supplemented to release the inhibition of the cellobiose product. Complete cellulase (CP-401 cell broth), as well as individual enzymes were characterized by standard BCA assays for total protein quantification. The dose responses of the enzyme mixtures were made by adding total known protein (calculated as added protein weight / glucan weight). The total protein levels tested were 0.73 and 3% (added protein weight / glucan weight). A dose response was measured at pH 5.0 at 55 ° C for 72 hours. The following enzyme combinations were used (in combination with BGL1):

to. CF-401 supernatant culture (Control; contains the 4 enzymes + GH61 proteins)

b.

50% (CBH1a + CBH2b) + 50% GH61f

C.

50% (CBH1a + CBH2b) + 50% GH61p

d.

50% (CBH1a + CBH2b) + 25% GH61f + 25% GH61p

and.

50% (CBH1a + CBH2b) + 16.6% GH61a + 16.6% GH61f + 16.6% GH61p

The reactions were quenched at 72 hours by the addition of 10 mM sulfuric acid. For glucose analysis, the samples were analyzed using an Agilent HPLC 1200 equipped with HPX-87H ion exclusion column (300 mM x 7.8 mM) with 5 mM H2SO4 as a mobile fas at a flow rate of 0.6 mL / min at 65 ° C. The glucose retention time was 9.1 minutes.

FIGURE 2 shows the results. The CBH1 and CBH2 cellulase enzymes were combined with several GH61 proteins. The control experiment (dotted line) was culture supernatant of CF-401 cells containing a plurality of cellulase enzymes and endogenous activity of GH61. The total protein load was added in the proportions specified in the figure.

These results establish that a minimum fixed enzyme is capable of achieving glucose levels similar to the control: specifically, CBH1 and CBH2 cellulases, combined with one or more of GH61f, GH61p and GH61a. All combinations generated higher glucose productions than the CP-401 culture broth. Thus, it is possible to achieve maximum theoretical conversions using a minimum set of enzymes. This also demonstrates that the minimum enzyme mixture used here has all the necessary components for the complete conversion of cellulose to glucose. However, it is contemplated that additional enzyme combinations will find use in saccharification processes.

Example 8. Synergy of GH61 activity with other CXP derived enzymes

This example describes an evaluation of GH61a for synergy with other enzymes derived from M. thermophila such as CBH1a, CBH2b and EG2.

The saccharification reactions were performed in a 110 g / L glucan load of pre-treated wheat straw at pH 5.0 at a temperature of 55 ° C at 950 rpm in a total volume of 95 µL in 96 deep well plates of high performance (HTP). 81 g / L xylose and 128 mM acetate were added to the pre-treated wheat straw. Excessive β-glucosidase (w / w glucan) was also supplemented to release the inhibition of the cellobiose product. Complete cellulase as well as individual enzymes were characterized by standard BCA assays for total protein quantification.

TABLE 6: Synergy of GH61a with enzymes derived from M. thermophila and enzyme mixtures

Enzymes CBH1a% CBH2b% CH61a% EG2% Production Load Level of complemented glucose synergy protein

with GH61a (total weight (g / L) protein added

/weight

glucan) CBH1a 0.39% -0.18% -0.57%> 15 gt; 1.6 CBH2b -0.30% 0.18% -0.48%> 35 gt; 1.3

EG2 -0.18% 0.20% 0.38%> 25> 1.2 CBH1a + CBH2b 0.39% 0.30% 0.18% -0.87%> 75> 1.7

CBH2b + EG2

- 0.30% 0.18% 0.20% 0.68% > 40 > 1.1

CBH1a + CBH2b

0.39% 0.30% 0.18% 0.20% 1.07% > 75 > 1.4

+ EG2

0.68% - 0.37% 0.20% 1.25% > 75 > 1.7

1.35%

1.20% 0.37% 0.20% 3.12% > 125 > 1.6

1.35%

1.20% 0.74% 0.20% 3.49% > 125 > 1.5

For an enzyme system comprising two enzymes A and B, the degree of synergy was calculated using the following formula:

Degree of Synergy =

The glucose production shown in the table is the production obtained from the combination of GH61 and enzymes. This is divided by glucose production measured separately for GH61 and the enzyme mixture (not shown) to quantify the synergy between the two.

15 The results show that GH61a has synergy with all enzyme systems derived from M. thermophila. For the complete conversion of cellulose to glucose, the presence of GH61a is beneficial.

Example 9. Use GH61a to reduce viscosity of pre-treated wheat straw

Purified GH61a from M. thermophila was evaluated to determine the enzymatic function and to evaluate any endo-glucanase type activity for reduction in cellulose chain length, thus allowing a reduction in viscosity.

GH61a alone or in combination with EG2 were tested for viscosity reduction in pre wheat straw

25 treated in glucan load of 75 g / L glucan and at pH 5.0, 50 ° C. The viscosity reduction tests were performed (30 minute cycle at 80 RPM) on an RVA-super4 viscometer (Newport Scientific, Australia) in a total weight of 21 g.

FIGURE 3 shows the results. The addition of 0.02% GH61a in relation to glucan showed approximately 2-3% reduction in viscosity at pH 5.50 ° C. In comparison, the addition of 0.01% M. thermophila EG2 relative to glucan showed approximately 19% viscosity reduction under the same conditions. With a combination of 0.02% GH61a and 0.01% EG2, the total viscosity reduction was approximately 21%.

SEQUENCES

5

Exemplary cellulase sequences

10

Precursor C1 BGL1: SEQ ID NO: 60

Precursor TaBGL (Thermoascus aurantiacus): SEQ ID NO: 61

Precursor CeIA BGL (Azospirillum irakense): SEQ ID NO: 62:

C1 CBH1a: SEQ ID NO: 63

C1 CBH2a precursor: SEQ ID NO: 64

M. thermophila Endoglucanase 2 (EG2): SEQ ID NO: 65

M. thermophila Beta-glucosidase (BG): SEQ ID NO: 66

M. thermophila Cellobiohydrolase Type 1a (Cbh1a): SEQ ID NO: 67

M. thermophila Cellobiohydrolase Type 2b (CBH2b): SEQ ID NO: 68

GH61a coding sequence

SEQ ID Nos: 32 to 59 appear in the formal Sequential Listing for this disclosure.

The use of GH61 proteins is not intended to be limited in any way to theory as its mode of action.

5 Theoretically, the production of the product can be increased by the action of the GH61 protein in the substrate, by interaction of the GH61 protein directly with one or more cellulase enzymes in a mixture, reducing the viscosity of the reaction mixture or by another mechanism . GH61 activity can be followed empirically using test methods described in this disclosure, without knowing the operating mechanism of the GH61 protein being used.

SPILL SEQUENCE

[0297]

<110gt; Codexis, Inc.

15 Rao, Kripa Clark, Louis Campopianio, Onorato Szabo, Lorand Torok, Janos Baidyaroy, Dipnath

<120gt; Use of Glycoside Hydrolase GH61 Family Proteins Facilitate cellulose processing

<130gt; 90834-818317

<150gt; US 61 / 375,788 25 <151gt; 2010-08-20

<160gt; 70

<170gt; PatentIn version 3.5

<210gt; one

<211gt; 342

<212gt; PRT

<213gt; Myceliophthora thermophila

<400gt; one

Claims (15)

 Claims 1. A composition comprising: a) a recombinantly produced GH61 protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 2 or fragments of SEQ ID NO: 2 that have GH61 activity; and b) cellulase enzymes that include at least one endoglucanase (EG), at least one β-glucosidase (BGL), at least one Type 1 cellobiohydrolase (CBH1), and at least one Type 2 cellobiohydrolase (CBH2).

2.

The composition of claim 1, wherein the GH61 protein: a) increases glucose production by at least 20% in a reaction where cellulose undergoes saccharification by a combination of enzymes comprising EG, BGL, CBH1 and CBH2, in comparison with the same reaction in the absence of the GH61 protein; b) comprises an amino acid sequence that is at least 85%, 90%, 95%, 97% or 99% identical to SEQ ID NO: 2; c) comprises SEQ ID NO: 2; or a fragment thereof that has GH61 activity.

3.

The composition of claim 1 or 2, wherein the cellulase enzymes are: a) recombinant; b) fungal enzymes, and / or c) of M. thermophila.

Four.

The composition of any one of claims 1 to 3, wherein the composition is obtained: a) expressing a gene encoding the GH61 protein in a host cell population, and expressing at least one gene encoding a cellulase enzyme in another population host cell; or b) expressing a gene that encodes the GH61 protein and at least one gene that encodes a cellulase enzyme in the same type of host cell.

5.

The composition of claim 4, wherein the gene encodes a GH61 protein comprising a heterologous signal peptide.

6.

A method for producing fermentable sugars from cellulosic biomass which comprises contacting a cellulosic substrate with a composition, culture medium or cell lysate containing a GH61 protein and cellulase enzymes, where:

a) GH61 protein comprises an amino acid sequence that is at least 89% identical to SEQ ID NO: 2 or a biologically active fragment thereof; Y b) cellulase enzymes include at least one endoglucanase (EG), at least one β-glucosidase (BGL), at less a Type 1 cellobiohydrolase (CBH1), and at least one Type 2 cellobiohydrolase (CBH2).

7.

The method of claim 6, wherein: a) before said contact, the substrate is pretreated; b), the composition, culture medium or cell lysate further comprises one or more enzymes selected from esterases, xylanases, hemicellulases, lipases, proteases, amylases, glucoamylases and combinations thereof; c) the cellulosic substrate is derived from wheat, wheat straw, sorghum, corn, rice, barley, sugarcane bagasse, herbs, rod grass, corn grain, corn cobs, corn fiber or a combination thereof , and where the substrate is optionally derived wheat straw; and / or d) the fermentable sugar comprises glucose.

8.

The method of claim 6 or 7, wherein the composition, culture medium or cell lysate comprises isolated GH61 and at least one or more of an isolated endoglucanase (EG), an isolated β-glucosidase (BGL), a Type 1 cellobiohydrolase isolated (CBH1), and / or an isolated Type 2 cellobiohydrolase (CBH2).

9.

A method for producing a final product from a cellulosic substrate, comprising: a) contacting the cellulosic substrate with a composition according to any of claims 1 to 5, whereby fermentable sugars are produced from the substrate; and b) contact the fermentable sugars with a microorganism in a fermentation to produce a final product.

10.

The method of claim 9, which is a simultaneous process of saccharification and fermentation.

eleven.

The method of claim 9, wherein the saccharification of the cellulosic substrate and said fermentation occur consecutively.

12.

A method of producing a final product from a cellulosic substrate, comprising:

a) obtaining fermentable sugars produced according to the method of any one of claims 6-8; and b) contact the fermentable sugars with a microorganism in a fermentation to produce a final product.

13.

The method of any one of claims 9 to 12, wherein the final product is an alcohol.

14.

The method of claim 13, wherein the alcohol is ethanol.

fifteen.

The method of any of claims 9 to 15, wherein the microorganism is a yeast.